Cancer is responsible for more than one in four deaths in the UK1, according to Cancer Research UK and one in five deaths in the USA, according to the Amercan Centers for Disease Control2. Prostate cancer is the second most common cancer in men in the US, with just under 300,000 new cases anticipated this year; one in eight men is expected to be diagnosed with it at some time during their life3.
In January, the American Cancer Society reported an increase in prostate cancer4, while Prostate Cancer UK report in the same month revealed growing disparities among different regions, where some receive the diagnosis too late5.
Palanisamy and Wei discussed the contribution made by their work, specifically the use of formalin-fixed paraffin-embedded (FFPE) preservation and preparation samples.
Can you give an overview of your research areas?
Palanisamy: I specialise in prostate cancer, cancer genomics, molecular cytogenetics, and molecular pathology. One key focus is to use formalin-fixed paraffin-embedded (FFPE) samples from patients with prostate cancer to learn more about the complexity of multifocal prostate cancer and its intra- and inter-tumor heterogeneity. I also look at genetic diversity in the incidence of molecular biomarkers by incorporating social, economic, and clinical information.
Why might there be a rise in the incidence of prostate cancer and variations across regions and countries?
Palanisamy: This is a big question, and more research is required. Based on the available information in the literature, the main reasons are complex social, economic, environmental, and behavioral reasons. Availability of resources for uniform access to health care and early detection screening are the limiting factors to assessing the exact reasons for determining the differences in the incidence in different populations.
As part of a Health Disparities Research Award, I discovered that African American men are 50% more likely to develop prostate cancer. Even more disturbing is that they are twice as likely to succumb to the disease as men of other races. We need to further apply innovative molecular approaches to better understand prostate cancer, especially as it relates to racial disparities. As part of my research, I used additional biomarkers to screen, and used the entire prostate tissue (wholemount) versus using sampling of a tumor foci. This helped us identify 260 samples out of 981 that returned positive for more than one biomarker each in a different focus within the prostate gland.
How can using FFPE tissue samples help with research into prostate cancer, its diagnosis and the development of treatment?
Palanisamy: Most surgical specimens are immediately fixed in formalin. Collection of fresh frozen tissues is time-consuming, requires dedicated staff, storage facilities and maintenance costs are expensive. Therefore, most of the biological specimens resulting from standard of care surgery are available in the form of formalin-fixed tissues. Recently, methods have been optimized to extract DNA. RNA and protein from FFPE tissues can be used to perform high throughput studies like comparative genomic hybridization, microarray, and next-generation sequencing. Many research centres have created resources to embed the entire prostate tissue collected after radical prostatectomy in formalin. This is an invaluable resource for an unbiased evaluation of highly heterogenous prostate cancer to assess the overall progression of the disease and develop novel approaches for early diagnosis.
What are the challenges of using FFPE samples and how can these be mitigated?
Wei: FFPE samples provide rich information to study human diseases such as cancer. However, due to the FFPE sample preparation methodology and storage conditions, several challenges are associated with using FFPE samples. Mainly, obtaining sufficient quantities of nucleic acid (e.g., DNA or RNA) of good quality remains difficult. Neutral buffered formalin preserves tissue histomorphology but causes crosslinking between proteins and nucleic acids, creates sequence artifacts in DNA, and contributes to reduced efficiency cDNA synthesis from RNA, which can lead to gene expression variations6.
In addition, formalin causes nucleic acid fragmentation, especially in RNA. This fragmentation can impair nucleic acid recovery and lead to the recognition of only short sequences (approximately 100-200 nucleotides).
When it comes to mitigations, first, the purification procedure must be highly efficient, enabling the recovery of as much usable analyte as possible. Many kits dedicated to DNA, RNA, or both extraction from FFPE sample are commercially available. The kit leverages gentle lysis to avoid further nucleic acid fragmentation, and degradation should be a major consideration when selecting a kit
Second, deparaffinization, which is the procedure that removes embedding paraffin from the tissue and has historically been conducted using the hazardous organic solvent xylene, can now be accomplished with kits using mineral oil. Mineral oil-based procedures have been shown to successfully eliminate labor-intensive steps, reduce hands-on time, and ultimately improve the yield and quality of extracted nucleic acid 7-8.
What have you learned from your research?
Palanisamy: Our research is focused on understanding the molecular heterogeneity of multifocal prostate cancer. Using prostate cancer-specific molecular markers, we have demonstrated the clonal heterogeneity of prostate cancer, which led to the identification of new molecular subsets of prostate cancer with improved clinical progression. Contrary to conventional approaches to looking at only the dominant or index tumor foci, our unbiased approach to evaluate the dominant and second tumor foci revealed an unprecedented molecular heterogeneity in prostate cancer in a racial disparity perspective.
Our research also focuses on identifying new molecular biomarkers and discovering novel genetic aberrations that can be used for cancer screening, non-invasive detection, and novel targeted therapeutics. We have also shown that some markers can be detected in patients' urine samples, enabling non-invasive cancer detection before invasive biopsy procedures.
Furthermore, we have identified new gene fusions which may be useful in the development of oncology drugs, and plan to run a larger cohort of FFPE samples to verify these findings.
Can using FFPE methodologies improve diagnosis and treatment in the future?
Wei: Yes. The research question and analytical technique may dictate the use of FFPE samples for particular research. For example, biobanked frozen samples of common cancers are likely to be available, but determinants such as associated treatments and availability of outcomes data may limit their application. On the other hand, clinical projects and trials that seek to establish the role of new biomarkers and associated targeted therapies usually rely on the retrieval of existing FFPE samples. For all practical purposes, microscopic lesions and rare tumors will exist primarily in FFPE samples9.
Next-generation sequencing (NGS) can provide in-depth analysis of short sequences, which makes it a powerful tool to use with nucleic acids isolated from FFPE tissue. Recently, researchers have started to leverage spatial multi-omics to locate and quantify DNA, RNA, and protein from FFPE samples in a 3-D fashion. It improves our understanding of tissue architecture and its molecular underpinning in health and disease. As this technique continues to evolve, we are committed to helping labs of all sizes embrace and optimize this workflow to further potentially life-saving research.
Dr. Nallasivam Palanisamy is an Associate Scientist at the Henry Ford Health System and Research Associate Professor at Michigan State University.
Dr. Han Wei is a Market Development Scientist at Beckman Coulter Life Sciences.
5. https://prostatecanceruk.org/about-us/news-and-views/2023/01/huge-north-south-divide-in- prostate-cancer-diagnoses
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7. Oba U, Kohashi K, Sangatsuda Y, Oda Y, Sonoda KH, Ohga S, Yoshimoto K, Arai Y, Yachida S, Shibata T, Ito T, Miura F. An efficient procedure for the recovery of DNA from formalin-fixed paraffin-embedded tissue sections. Biol Methods Protoc. 2022 Jul 26;7(1):bpac014. doi: 10.1093/biomethods/bpac014. PMID: 35937639; PMCID: PMC9351614.
8. van Maldegem F, de Wit M, Morsink F, Musler A, Weegenaar J, van Noesel CJ. Effects of processing delay, formalin fixation, and immunohistochemistry on RNA Recovery From Formalin-fixed Paraffin-embedded Tissue Sections. Diagn Mol Pathol. 2008 Mar;17(1):51-8. doi: 10.1097/PDM.0b013e31814b8866. PMID: 18303406
9. Gaffney EF, Riegman PH, Grizzle WE, Watson PH. Factors that drive the increasing use of FFPE tissue in basic and translational cancer research. Biotech Histochem. 2018;93(5):373- 386. doi: 10.1080/10520295.2018.1446101. Epub 2018 Aug 16. PMID: 30113239