Economic assessment of NGS testing workflow for NSCLC in a healthcare setting.

Background: The molecular diagnostic and therapeutic pathway of Non-Small Cell Lung Cancer (NSCLC) stands as a successful example of precision medicine. The scarcity of material and the increasing number of biomarkers to be tested have prompted the routine application of next-generation-sequencing (NGS) techniques. Despite its undeniable advantages, NGS involves high costs that may impede its broad adoption in laboratories. This study aims to assess the detailed costs linked to the integration of NGS diagnostics in NSCLC to comprehend their financial impact. Materials and methods: The retrospective analysis encompasses 210 cases of early and advanced stages NSCLC, analyzed with NGS and collected at the IRCCS San Gerardo dei Tintori Foundation (Monza, Italy). Molecular analyses were conducted on FFPE samples, with an hotspot panel capable of detecting DNA and RNA variants in 50 clinically relevant genes. The economic analysis employed a full-cost approach, encompassing direct and indirect costs, overheads, VAT (Value Added Tax). Results: We estimate a comprehensive cost for each sample of €1048.32. This cost represents a crucial investment in terms of NSCLC patients survival, despite constituting only around 1% of the expenses incurred in their molecular diagnostic and therapeutic pathway. Conclusions: The cost comparison between NGS test and the notably higher therapeutic costs highlights that the diagnostic phase is not the limiting economic factor. Developing NGS facilities structured in pathology networks may ensure appropriate technical expertise and efficient workflows.

Nephrosphere-Derived Cells Are Induced to Multilineage Differentiation when Cultured on Human Decellularized Kidney Scaffolds.

In end-stage chronic kidney disease, the option of organ transplantation is limited because of the scarce availability of kidneys. The combination of stem cell research, regenerative medicine, and tissue engineering seems a promising approach to produce new transplantable kidneys. Currently, the possibility to repopulate naturally obtained scaffolds with cells of different sources is advancing. Our aim was to test, for the first time, whether the nephrosphere (NS) cells, composed by renal stem/progenitor-like cells, were able to repopulate different nephron portions of renal extracellular matrix scaffolds obtained after decellularization of human renal tissue slices. Our decellularization protocol enabled us to obtain a completely acellular renal scaffold while maintaining the extracellular matrix structure and composition in terms of collagen IV, laminin, and fibronectin. NS cells, cultured on decellularized renal scaffolds with basal medium, differentiated into proximal and distal tubules as well as endothelium, as highlighted by histology and by the specific expression of epithelial cytokeratin 8.18, proximal tubular CD10, distal tubular cytokeratin 7, and endothelial von Willebrand factor markers. Endothelial medium promoted the differentiation toward the endothelium, whereas epithelial medium promoted the differentiation toward the epithelium. NS cells seem to be a good tool for scaffold repopulation, paving the way for experimental investigations focused on whole-kidney reconstruction.

Multiplex Staining by Sequential Immunostaining and Antibody Removal on Routine Tissue Sections.

Multiplexing, labeling for multiple immunostains in the very same cell or tissue section in situ, has raised considerable interest. The methods proposed include the use of labeled primary antibodies, spectral separation of fluorochromes, bleaching of the fluorophores or chromogens, blocking of previous antibody layers, all in various combinations. The major obstacles to the diffusion of this technique are high costs in custom antibodies and instruments, low throughput, and scarcity of specialized skills or facilities. We have validated a method based on common primary and secondary antibodies and diffusely available fluorescent image scanners. It entails rounds of four-color indirect immunofluorescence, image acquisition, and removal (stripping) of the antibodies, before another stain is applied. The images are digitally registered and the autofluorescence is subtracted. Removal of antibodies is accomplished by disulfide cleavage and a detergent or by a chaotropic salt treatment, this latter followed by antigen refolding. More than 30 different antibody stains can be applied to one single section from routinely fixed and embedded tissue. This method requires a modest investment in hardware and materials and uses freeware image analysis software. Multiplexing on routine tissue sections is a high throughput tool for in situ characterization of neoplastic, reactive, inflammatory, and normal cells.

Antigen Masking During Fixation and Embedding, Dissected.

Antigen masking in routinely processed tissue is a poorly understood process caused by multiple factors. We sought to dissect the effect on antigenicity of each step of processing by using frozen sections as proxies of the whole tissue. An equivalent extent of antigen masking occurs across variable fixation times at room temperature. Most antigens benefit from longer fixation times (>24 hr) for optimal detection after antigen retrieval (AR; for example, Ki-67, bcl-2, ER). The transfer to a graded alcohol series results in an enhanced staining effect, reproduced by treating the sections with detergents, possibly because of a better access of the polymeric immunohistochemical detection system to tissue structures. A second round of masking occurs upon entering the clearing agent, mostly at the paraffin embedding step. This may depend on the non-freezable water removal. AR fully reverses the masking due both to the fixation time and the paraffin embedding. AR itself destroys some epitopes which do not survive routine processing. Processed frozen sections are a tool to investigate fixation and processing requirements for antigens in routine specimens.

α-1-Antitrypsin detected by MALDI imaging in the study of glomerulonephritis: Its relevance in chronic kidney disease progression.

Idiopathic glomerulonephritis (GN), such as membranous glomerulonephritis, focal segmental glomerulosclerosis (FSGS), and IgA nephropathy (IgAN), represent the most frequent primary glomerular kidney diseases (GKDs) worldwide. Although the renal biopsy currently remains the gold standard for the routine diagnosis of idiopathic GN, the invasiveness and diagnostic difficulty related with this procedure highlight the strong need for new diagnostic and prognostic biomarkers to be translated into less invasive diagnostic tools. MALDI-MS imaging MALDI-MSI was applied to fresh-frozen bioptic renal tissue from patients with a histological diagnosis of FSGS (n = 6), IgAN, (n = 6) and membranous glomerulonephritis (n = 7), and from controls (n = 4) in order to detect specific molecular signatures of primary glomerulonephritis. MALDI-MSI was able to generate molecular signatures capable to distinguish between normal kidney and pathological GN, with specific signals (m/z 4025, 4048, and 4963) representing potential indicators of chronic kidney disease development. Moreover, specific disease-related signatures (m/z 4025 and 4048 for FSGS, m/z 4963 and 5072 for IgAN) were detected. Of these signals, m/z 4048 was identified as α-1-antitrypsin and was shown to be localized to the podocytes within sclerotic glomeruli by immunohistochemistry. α-1-Antitrypsin could be one of the markers of podocyte stress that is correlated with the development of FSGS due to both an excessive loss and a hypertrophy of podocytes.

Disaccharides Protect Antigens from Drying-Induced Damage in Routinely Processed Tissue Sections.

Drying of the tissue section, partial or total, during immunostaining negatively affects both the staining of tissue antigens and the ability to remove previously deposited antibody layers, particularly during sequential rounds of de-staining and re-staining for multiple antigens. The cause is a progressive loss of the protein-associated water up to the removal of the non-freezable water, a step which abolishes the immunoavailability of the epitope. In order to describe and prevent these adverse effects, we tested, among other substances, sugars, which are known to protect unicellular organisms from freezing and dehydration, and stabilize drugs and reagents in solid state form in medical devices. Disaccharides (lactose, sucrose) prevented the air drying-induced antigen masking and protected tissue-bound antigens and antibodies from air drying-induced damage. Complete removal of the bound antibody layers by chemical stripping was permitted if lactose was present during air drying. Lactose, sucrose and other disaccharides prevent air drying artifacts, allow homogeneous, consistent staining and the reuse of formalin-fixed, paraffin-embedded tissue sections for repeated immunostaining rounds by guaranteeing constant staining quality in suboptimal hydration conditions.

A 2-Step Laemmli and Antigen Retrieval Method Improves Immunodetection.

Detection by immunohistochemistry of antigens relies on reproducibly optimal preanalytical and analytical variables such as fixation conditions, antigen retrieval (AR), and the resolutive power of the detection system. There is a need to improve immunodetection on routinely fixed and embedded material, particularly for scarcely represented but relevant antigens. We devised a 2-step method and applied it to a panel of antigens of common use for diagnosis, prognosis, individualized therapy use, or research. The first step consists of a 10 minutes. Incubation at 95°C with a modified Laemmli extraction buffer. This was followed by a traditional AR method. Detection of the vast majority of antigens was improved over a simple AR with preservation of tissue integrity, as shown by quantitative image analysis. The mechanism underlying the improved detection may be controlled denaturation followed by heat-mediated retrieval, a method we dubbed "antigen relaxing" and which will improve routine detection of scarce antigens in formalin-fixed, paraffin-embedded material.

Epitope recognition in the human-pig comparison model on fixed and embedded material.

The conditions and the specificity by which an antibody binds to its target protein in routinely fixed and embedded tissues are unknown. Direct methods, such as staining in a knock-out animal or in vitro peptide scanning of the epitope, are costly and impractical. We aimed to elucidate antibody specificity and binding conditions using tissue staining and public genomic and immunological databases by comparing human and pig-the farmed mammal evolutionarily closest to humans besides apes. We used a database of 146 anti-human antibodies and found that antibodies tolerate partially conserved amino acid substitutions but not changes in target accessibility, as defined by epitope prediction algorithms. Some epitopes are sensitive to fixation and embedding in a species-specific fashion. We also find that half of the antibodies stain porcine tissue epitopes that have 60% to 100% similarity to human tissue at the amino acid sequence level. The reason why the remaining antibodies fail to stain the tissues remains elusive. Because of its similarity with the human, pig tissue offers a convenient tissue for quality control in immunohistochemistry, within and across laboratories, and an interesting model to investigate antibody specificity.

Elution of High-affinity (>10-9 KD) Antibodies from Tissue Sections: Clues to the Molecular Mechanism and Use in Sequential Immunostaining.

Inconsistent results obtained with published methods for the elution of antibodies from tissue sections prompted the assessment of both old and new methods in combination with monoclonal rabbit antibodies of known, increased affinity (above 1×10(-9) KD). We tested an acidic (pH 2) glycine buffer, a 6 M urea hot buffer and a 2-Mercaptoethanol, SDS buffer (2-ME/SDS). Some antibodies were not removed by the glycine pH 2 or 6 M urea hot buffers, indicating that antibodies survive much harsher conditions than previously believed. We found that the elution is dependent upon the antibody affinity and is reduced by species-specific crosslinking via a dimeric or Fab fragments of a secondary antibody. The high affinity bond of exogenous streptavidin with the endogenous biotin can be removed by 6 M urea but not by the other buffers. 2-ME/SDS buffer is superior to glycine pH 2 and 6 M urea hot elution buffers for all antibodies because of its irreversible effect on the structure of the antibodies. It also has a mild retrieving effect on some antigens present on routinely treated sections and no detrimental effect on the immunoreactivity of the tissue. Therefore, 2-ME/SDS buffer is the method of choice to perform multiple rounds of immunostaining on a single routine section.