Search

The PAM50 gene expression subtypes and the associated risk of recurrence (ROR) score are used to predict the risk of recurrence and the benefits of adjuvant therapy in early-stage breast cancer. The Prosigna assay includes the PAM50 subtypes along with their clinicopathological features, and is approved for treatment recommendations for adjuvant hormonal therapy and chemotherapy in hormone-receptor-positive early breast cancer. The Prosigna test utilizes RNA extracted from macrodissected tumor cells obtained from formalin-fixed, paraffin-embedded (FFPE) tissue sections. However, RNA extracted from fresh-frozen (FF) bulk tissue without macrodissection is widely used for research purposes, and yields high-quality RNA for downstream analyses. To investigate the impact of the sample preparation approach on ROR scores, we analyzed 94 breast carcinomas included in an observational study that had available gene expression data from macrodissected FFPE tissue and FF bulk tumor tissue, along with the clinically approved Prosigna scores for the node-negative, hormone-receptor-positive, HER2-negative cases (n = 54). ROR scores were calculated in R; the resulting two sets of scores from FFPE and FF samples were compared, and treatment recommendations were evaluated. Overall, ROR scores calculated based on the macrodissected FFPE tissue were consistent with the Prosigna scores. However, analyses from bulk tissue yielded a higher proportion of cases classified as normal-like; these were samples with relatively low tumor cellularity, leading to lower ROR scores. When comparing ROR scores (low, intermediate, and high), discordant cases between the two preparation approaches were revealed among the luminal tumors; the recommended treatment would have changed in a minority of cases.

This study aimed to compare the performance of MilliSect dissection and manual dissection. Twenty-five formalin-fixed paraffin-embedded (FFPE) breast cancer tissue blocks were selected for comparison. Specific areas of interest (AOIs) in invasive carcinoma on tissue sections were transferred to dissection slides by manual macrodissection or the MilliSect instrument. The comparison criteria were 1) the time required for dissection; 2) RNA concentration and purity; 3) RNA quantity of 5 housekeeping genes (by RT-qPCR); and 4) ER, PR, HER2, Ki-67 and recurrence score (RS) values (by the 21-gene assay). Then, tumor-adjacent tissues, including fibrocollagenous and epithelial tissues, from the same selected tissue blocks of 8 of 25 patients were scraped using the mesodissection method, and their RS values were assessed to evaluate the influence of tumor-adjacent tissues on the target AOIs. Ultimately, 4 AOIs of invasive ductal carcinoma (IDC) from 1 tissue block of another 4 patients with lymph node (LN) metastases each, LN tissue and a mixture of IDC and LN tissue from the other tissue block of the same 4 patients were mesodissected to evaluate the influence of infiltrating lymphocyte levels on the RS values of AOIs. In our experience, the MilliSect instrument, which provides process management documentation, required more time than manual macrodissection (on average, approximately 9.1 min per sample versus 5.8 min per sample, respectively). The RNA yield and quality of the dissected tissues were comparable for the 2 methods. However, the tumor-adjacent tissues of the AOIs may influence the RS to some extent. Tumor-infiltrating lymphocytes (TILs) can dramatically increase RSs, far exceeding the influence of tumor-adjacent fibrocollagenous and epithelial tissues. In conclusion, MilliSect mesodissection is comparable to manual dissection. This mesodissection tool may facilitate AOI alignment and the dissection process for the 21-gene RS assay. Samples whose adjacent tissues are intermixed with TILs warrant special attention.

Somatic mutations in patient tumor DNA samples can be readily detected based on mass spectrometry. The MassARRAY system is a high-throughput matrix-assisted laser desorption time-of-flight (MALDI) mass spectrometer for detection of nucleic acids. The technique is based on single-nucleotide base extension. A series of PCR assays amplify specific DNA regions of interest harboring mutations. A third primer is then introduced into the reaction which corresponds to the DNA template immediately in front of the mutation site. A final round of PCR is then performed using mass-modified nucleotides. These nucleotides are designed so that no additional bases can be added to the extension primer (terminating bases) after a single-base extension and are mass modified to exaggerate mass differences between nucleotides allowing easier identification by mass spectrometry.The sequences of the extension primer and possible extension products (wild type and mutations) are known; therefore, it is possible to calculate their mass. The mass spectrometer can identify the mass peaks for each assay and identify those with mutations (multiple peaks). The technique was originally designed to screen multiple single-nucleotide polymorphisms (SNPs) in a large number of specimens. A SNP in the coding region of DNA that alters the gene and subsequent protein expression is considered a mutation. Mutations often occur in genes whose protein product is in a key signaling pathway and/or drug target. Rationale treatment options can be designed based upon the presence or absence of these mutations. In this chapter, we describe the process for detection of somatic mutations in DNA extracted from formalin-fixed paraffin-embedded (FFPE) material.

Background: Data generated by next-generation sequencing technologies have a pivotal role in precision medicine. These high-throughput techniques are preferentially performed on fresh tissue, but there is an increasing need for protocols adapted to materials derived from formalin-fixed, paraffin-embedded tissue and cytology specimens., Methods: The aim of this work was to show that cytological material collected from archival smears processed for routine diagnoses could be used for massively parallel sequencing and array-based genomic analysis for further studies., Results: As a proof of concept, data obtained from May-Grünwald Giemsa- and Diff-Quik-stained archival smears were shown to be in keeping with those obtained from matched frozen controls., Conclusions: The quality of DNA extracted from routinely processed smears is compatible with the multitargeted sequencing of a large series of genes of interest with methods such as array-based genomic analysis and whole-exome sequencing., (© 2015 American Cancer Society.)

Molecular genetic testing on formalin fixed, paraffin embedded (FFPE) tumors frequently requires dissection of tumor from tissue sections mounted on glass slides. In a process referred to as "manual macrodissection," the pathologist reviews an H&E stained slide at the light microscope and marks areas for dissection, and then the laboratory performs manual dissection from adjacent sections without the aid of a microscope, using the marked reference H&E slide as a guide. Manual macrodissection may be inadequate for tissue sections with low tumor content. We compared manual macrodissection to a new method, digitally guided microdissection, on a series of 32 FFPE pancreatic cancer samples. KRAS hotspot mutation profiling was performed using the Sequenom MassARRAY system (Agena Bioscience). Digitally guided microdissection was performed on multiple smaller areas of high tumor content selected from within the larger areas marked for manual macrodissection. The KRAS mutant allele fraction and estimated neoplastic cellularity were significantly higher in samples obtained by digitally guided microdissection (p < 0.01), and 7 of the 32 samples (22%) showed a detectable mutation only with digitally guided microdissection. DNA quality and yield per cubic millimeter of dissected tissue were similar for both dissection methods. These results indicate a significant improvement in tumor content achievable with digitally guided microdissection., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Formalin fixation, duration of tissue storage and tissue enrichment techniques can affect DNA methylation yield but these effects have not been quantitatively measured. The aim is to investigate the relative impact of these conditions on DNA methylation in rectal cancer., Methods: 10 rectal cancers with matched undissected fresh frozen tissues, laser capture microdissected (LCM) formalin-fixed paraffin-embedded (FFPE) tissues, manual macrodissected FFPE tissues, adjacent normal mucosa and stromal tissues were analysed for APC and LINE-1 methylation using bisulphite pyrosequencing., Results: FFPE cancer tissues, which had been stored for at least 4 years showed similar APC and LINE-1 methylation changes to matched fresh frozen cancer tissues. Laser capture microdissection did not increase the degree of methylation detected compared to manual macrodissection. Analysis of stromal tissues showed that they had undergone significant methylation changes compared to adjacent macroscopically normal mucosa, but not to the same extent as cancer tissues., Conclusion: Reliable DNA methylation results can be obtained from FFPE rectal cancer tissues, which have been in long-term storage. Because only minor differences in methylation between macrodissected and LCM cancer tissues were found, our results do not support the routine use of LCM to enrich for cancer cells for DNA methylation studies., (© 2013.)