enhancing-tumor-content-through-tumor-macrodissection

Enhancing Tumor Content through Tumor Macrodissection

The presence of contaminating non-tumor tissues in formalin-fixed paraffin-embedded (FFPE) tissues can greatly undermine genomic studies. Herein we ...

manual-construction-tissue-microarray-using-tape-method-handheld

Manual Construction of a Tissue Microarray using the Tape Method and a Handheld Microarrayer

The tissue microarray (TMA) is an important research tool in which many formalin fixed paraffin embedded (FFPE) samples can be represented in a single ...

Mayo Clinic

Tissue microarrays (TMAs) are important tools to conserve precious tissue resources from increasingly smaller biopsies and to control experimental costs and variation across sample sets. The quality assurance assessment of TMA materials created at centralized biobanks has not been standardized. Herein, we outline 2 processes for the construction of TMAs ("recipient block" and "tape" methods) and the associated preconstruction quality control measures (pathology review, protein and RNA assessment, map creation, and storage conditions) developed by the AIDS Cancer Specimen Resource (ACSR) Network's Science and Technology Core. These steps provide a suggested framework for quality assessment that allows end-users, receiving materials from tissue banks, confidence in their experimental results.

Recommendations for Tissue Microarray Construction and Quality Assurance.

Diffuse large B-cell lymphoma (DLBCL) is up to 17-fold more likely to occur, follows a more aggressive clinical course and frequently presents at advanced stages in HIV infected (+) individuals compared to HIV negative (-) individuals. However, the molecular pathology underpinning the clinical features of DLBCL in HIV(+) patients relative to the general population is poorly understood. We performed a retrospective study examining the transcriptional, genomic and protein expression differences between HIV(+) and HIV(-) germinal center B-cell (GCB) DLBCL cases using digital gene expression analysis, array comparative genomic hybridization (CGH) and immunohistochemistry (IHC). Genes associated with cell cycle progression (CCNA2, CCNB1, CDC25A, E2F1), DNA replication (MCM2, MCM4, MCM7) and DNA damage repair, including eight Fanconi anemia genes (FANCA, FANCD1/BRCA2, FANCE, FANCG, FANCR/RAD51, FANCS/BRCA1, FANCT/UBE2T, FANCV/MAD2L2), were significantly increased in HIV(+) GCB-DLBCL tumors compared to HIV(-) tumors. In contrast, genes associated with cell cycle inhibition (CDKN1A, CDKN1B) as well as apoptosis regulating BCL2 family members (BCL2, BAX, BIM, BMF, PUMA) were significantly decreased in the HIV(+) cohort. BCL2 IHC confirmed this expression. Array CGH data revealed that HIV(+) GCB-DLBCL tumors have fewer copy number variations than their HIV(-) counterparts, indicating enhanced genomic stability. Together, the results show that HIV(+) GCB-DLBCL is a distinct molecular malignancy from HIV(-) GCB-DLBCL; with an increased proliferative capacity, confirmed by Ki67 IHC staining, and enhanced genomic stability, the latter of which is likely related to the enhanced expression of DNA repair genes.

Enhanced DNA repair and genomic stability identify a novel HIV-related diffuse large B-cell lymphoma signature.

 Formalin-fixed, paraffin-embedded (FFPE) tissues are a valuable resource for clinical and basic science research. Paraffin blocks and the resulting unstained sections (USS) are often stored for years before being used. Previous studies have evaluated the effects of time, temperature, humidity, and inert gases on preservation of USS; however, no study has examined all four variables together.  In the current work, we prospectively and blindly assessed time points from 0 to 24 months, room versus refrigerated temperature, and presence of a desiccant and/or nitrogen atmosphere on a variety of benign and malignant tissues from North America and Africa. End points included immunohistochemistry (IHC),  hybridization (ISH), extracted RNA and DNA quantity and quality, and messenger RNA performance in a novel, multiplexed digital gene expression profiling assay of both housekeeping and tumor-specific genes.  We found that using current methods of antigen retrieval, staining, and extraction, the end points of IHC, ISH, RNA, and DNA were well preserved under the various conditions tested, with implications that pre-embedding factors contribute to variability in subsequent tissue integrity. We also document that spectrophotometric estimations of nucleic acid concentrations were in general estimated to be higher than with fluorimetric methods, which may be pertinent to end assay development. We further describe a new multiplex assay, the PlexSet digital gene expression assay, suitable for evaluating RNA quality in FFPE tissues.  Altogether, these results may provide helpful guidance with regard to approaches for long-term storage conditions for USS.

Assessment of 2-Year Storage Conditions on Protein, RNA, and DNA in Unstained Human Tissue Sections, Including a Novel Multiplex Digital Gene Expression Profiling Method with Implications for Biobanking.

 The AIDS and Cancer Specimen Resource (ACSR) is a network of four regional biospecimen repositories and a technical core in the United States and South Africa. Its mission is to acquire, store, and distribute HIV-associated malignancy specimens and related clinical data to support translational research. At the outset of the COVID-19 pandemic, it became apparent that existing ACSR Standard Operating Procedures (SOPs) were not sufficient to ensure long-term maintenance and integrity of inventories during periods of extended shutdown. The ACSR needed an administrative SOP for situations pertaining to epidemics/pandemics. The ACSR Quality Working Group (QWG), comprised of representatives from each of the five ACSR sites and an external member who directs a large university medical center biorepository, addressed the issue.  To understand the individual problems the sites faced, questions were developed to query each of the six QWG sites' contingency plans to cover this type of emergency, the amount of work allowed onsite and by whom, the challenges sites experienced, and the lessons learned to assist with future similar situations, while remaining consistent with the existing IRB protocols.  Reported challenges spanned all activities of classical biobanks and differed within the geographical locations of the sites and the local COVID-19 infection rate. Review of the responses to the questions revealed that the general shutdown of society external to the biorepositories presented them with a homogeneous collection of problems, limitations, and needs. This led to creating an SOP that addresses planning for pandemic emergencies, scaling down of activities, shutting down, and reopening plans.  The ACSR QWG sites now have a structured response SOP for their sites, including guidance on how to develop and implement an emergency shutdown and reopening plan. The complete SOP is publicly available on the ACSR website.

Lee Wisner

Department of Research

Mayo Clinic

Enhancing Tumor Content through Tumor Macrodissection

Manual Construction of a Tissue Microarray using the Tape Method and a Handheld Microarrayer

Lee Wisner

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Research

Mayo Clinic

Enhancing Tumor Content through Tumor Macrodissection

Manual Construction of a Tissue Microarray using the Tape Method and a Handheld Microarrayer

Department of Research