5037BMS H&E Assessment of Clear Cell Renal Cell Carcinoma Lab Report Answer

Introduction

Cancer arising from the resident cells i.e. renal epithelial cells located in the renal tubule lining is referred to as renal cell carcinoma (RCC). It is of majorly of three distinct types depending upon the histological pattern with the more commonly occurring being clear cell renal cell carcinoma (ccRCC) (Hsieh et al., 2017). On microscopic examination, the ccRCC will develop vacuolated cytoplasm which is why they are called ccRCC. The pathophysiology of RCC more precisely ccRCC is influenced by varied genetic alterations. The review conducted by Nabi et al., (2018) highlights that the deletion of chromosome 3's short arm i.e. p arm is associated with nearly 95% of ccRCC cases. Sporadic and genetic are the further two subtypes of ccRCC. Genes such as Von Hippel-Lindau (VHL), Protein polybromo-1 gene (PBRM-1), and BRAC1- associated protein-1 (BAP-1) are the major genes that influence the pathogenesis of ccRCC. A type of gene associated with tumor suppression, VHL plays an imperative role in the development of ccRCC. The inactivation associated with this gene is the causative factor for the incidences of ccRCC. Hypoxia‐inducible transcription factors (HIFs) are activated as a consequence of the inactivation of VHL. The genes PBRM1 and BAP1 are also near VHL genes which are also associated with the loss of 3p genes leading to the oncogenesis of ccRCC (Signoretti et al., 2018). Epithelial-to-mesenchymal transition (EMT) is defined as the process of acquiring mesenchymal composition by epithelial cells. EMT aids in the development of cancer progression and helps in local invasion and remote metastasis. EMT pathway is also associated with the loss of epithelial cells' cell-to-cell adhesion properties. The activation of transcription factors like SNAIL1, ZEB1, etc. is associated with processes advocating the evolution of EMT. The factors are associated with the expression of certain proteinases that further impacts the processes related to EMT progression in ccRCC. These enzymes like MMP2 and MMP9 cause delayed identification of ccRCC (Landolt et al., 2017). Adhesion molecules like cadherin may be associated with the progression of tumorigenesis. Epithelial cell adhesion molecules (EpCAM) expresses largely in ccRCC. These adhesion molecules are also associated with the proliferation of tumors in RCC (Tolmachev et al., 2022). These molecules are also known as the diagnostic marker for the RCC. The following report highlights the case of a patient who is depicting symptoms of lower back pain, decreased appetite, and the presence of blood in the urine. The physicians believe that the patient is presenting symptoms of either cancer or kidney disease. The present report aims to confirm the presence of cancer/ kidney diseases in a patient showing the abovementioned symptoms in laboratory settings. The report also highlights the justification behind using the current lab investigation methods for the scope of the present case study.

Justification of the methods

Lab Session 1: H&E staining

Histologic techniques can be used to assess the histological structures of diverse human tissues. The H&E staining can be employed for the detection of ccRCC. The major aim of the H&E staining is to visualize changes in the morphology of the cytoplasm and the nuclei (Park et al., 2023). For the current experiment, the von kossa silver stain method has been employed.

The research design for the Von kossa silver stain method is as followed:

Mouse kidney samples were used and two slides named '7' and 'control' was used. The aim was to identify the location of the alkaline phosphatase enzyme in the mouse kidney sections. Then dewaxing was done and histoclear was used as the clearing agent. Rehydration with different concentrations of alcohol was done. Then the slide other than the control will be put into the substrate and then will be treated with histochemical stain. Then after incubation will be treated with H&E stain along with the control. The position where the calcium is reduced by the silver nitrate substrate will be visualized in black.

Lab Session 2: Immunophenotyping and Flow Cytometry

Immunophenotyping procedures are utilized to determine the presence of a cell population of interest from the heterogeneous population. Flow cytometer Immunophenotyping assesses intracellular antigens visualized by cancerous cells (DiGiuseppe & Wood, 2019). Flow cytometer asses the light emitted by fluorescently labeled antibodies to detect antigenic markers. For the current experiment, different fluorochrome-labeled antibody to detect renal carcinoma cells. Sample from the healthy and the patient is taken for detection. Two mixes of antibodies conjugated with PerCP and FITC having distinct markers present in renal cell carcinoma cells are utilized for detection purposes. Once the sample is mixed with the antibody mix, the solution is taken into the flow cytometer.

Lab Sessions 3&4: Sandwich ELISA and the research design for the current experiment

ELISA is a basic immunoassay test that is employed for the detection and quantification of proteins, antibodies, or antigens (Alhajj & Farhana, 2022). The procedure will be carried out in two distinct lab sessions with one session devoted to plating and this plate will be utilized for session 2. The source of antigen is the serum samples from the healthy and diseased patient serum. The first step involves the capturing of the primary antibody on the surface of the 96-well microtiter plate and the preparation of the standard solution, in tubes 1 to 7 with tube 8 being the control. Followed by the incubation of the primary antibody the serum samples are added which are the source of the antigen. Following the incubation a detection antibody with a streptavidin-HRP conjugate for detection purposes. A substrate was added which reacts with the enzyme conjugate and produces color which can be read ta 450nm in a spectrophotometer.

Results

H&E staining results

After staining with the AgNO₃ and H&E stain with the healthy and mice samples, the cancerous cells were depicted as black spots due to the oxidation of calcium with silver nitrate. After examining the results under the microscope it was perceived that the in the AgNO₃ + H&E stain (part C) of the patient sample black spots were visible which demonstrated abnormal calcium deposits. As the result, the black spots are visible at the proximal convoluted tubule regions (PCT). The PCT is demonstrated by the thick border and narrow lumen. This marks the location of calcium phosphate which is formed after the enzyme alkaline phosphatase splits into phosphate and reacts with calcium. Due to the presence of more calcium phosphate is formed and can be easily detected. Whereas part D demonstrates the histopathology H&E stain of the mouse kidney sample in which the nuclei are purple and the cytoplasm is pink. Figure D depicts the clear cell carcinoma due to the vacuolated cytoplasm of PCT due to the presence of glycogen and lipid.

Sandwich ELISA results

The major aim of the experiment was to detect the presence of high levels of cytokine interleukin-6 (IL-6) in the patient and healthy serum samples. Serum was assessed to check for the levels of IL-6.

After interpreting the results of the ELISA test it was visualized that the concentration of target protein was found somewhere at 0.94 pg/ml in the patient sample. This is the concentration of IL-6 in the patient sample. This depicts that the IL-6 concentration may be depictive of the RCC. Increased cytokine levels may be indicative of the prognosis of renal cell carcinoma in the patient sample.

Immunophenotyping Results

Flow cytometry immunophenotyping was used to analyze the markers present in the cancerous cells. Two fluorophores PerCP (emitting red fluorescence) and FITC-A (emitting blue fluorescence) are used to detect the cell populations. After analysis of the histograms obtained after the flow cytometer procedure of the healthy and patient sample distinct results were obtained. In the histogram for the healthy sample for mix 1, the population of cells was visualized in a lower left compartment which depicts the negativity for both the antibody present in mix one. Similar results are obtained for mixing 2 antibodies. Whereas for the patient sample, the cell population lies in the lower right quadrant and depicts increased green fluorescence i.e. positivity for CD10 which may be indicative of ccRCC. The mix2 histogram results display the cell population lying in the lower left compartment and a few of the population is present in an upper left compartment which may indicate the presence of ccRCC and confirms the presence of CD44 and CD105. 

Figure 2: Demonstrates the dot plots for the healthy sample with two different antibody mixtures.

Figure 3: Demonstrates the dot plots for the patient sample with two different antibody mixtures.

Discussion

Renal cancer or ccRCC has its origin in PCT and is depicted by vacuolated cytoplasm due to the accumulation of lipids and glycogen. The ccRCC is demonstrated by having a clear cytoplasm. The assignment aimed to encircle the experimental procedures that can be employed to assess the positivity of RCC more precisely ccRCC. After analyzing the results obtained from the laboratory sessions many key findings were highlighted. Von Koss method of staining was utilized in lab session 1 to visualize the areas where calcium phosphate was present, followed by H&E staining which gives the histological distinction of the kidney sections of the normal and diseased samples. Increased levels of alkaline phosphatase levels are determinants of chronic kidney disorders (Bover et al., 2018). The test was employed to analyze the concentration of the alkaline phosphates enzyme. The silver cations give a yellow color after the reaction with calcium ions which then develops black color via the light reaction hence this property is exploited to visualize calcium deposits (Schneider, 2021). Hyperglycemia is visualized in renal cell carcinomas. As a result, the black spots are visible at the proximal convoluted tubule regions (PCT). The PCT is demonstrated by the thick border and narrow lumen. This marks the location of calcium phosphate which is formed after the enzyme alkaline phosphatase splits into phosphate and reacts with calcium. H&E staining demonstrated the vacuolated cytoplasm which demonstrates the presence of RCC. In lab session 2 flow cytometry Immunophenotyping procedures were carried out to test the presence of RCC more precisely ccRCC. This technique is utilized to measure the protein levels within a cell population by utilizing a fluorescently labeled antibody (Herold & Mitra, 2022). Gating is imperative while data analysis as it aids in eliminating the unwanted cell population. The histogram is divisible into 4 distinct quadrants and each quadrant demonstrates the presence of a different cell population. Two different antibody mixes were utilized. The antibody mix 1 consisted of CK7 and CD10. These both are glycoproteins present on the cell surfaces and in the presence of antigen show a reaction and can confer the presence of ccRCC as CK7 is negative in this condition (Farber et al., 2017). CD10 may be indicative of ccRCC. Whereas mix 2 consists of CD44 and CD105 as the glycoproteins. CD44 and CD105 are the diagnostic markers for ccRCC malignancy and both are the most widely recognized markers (Fiedorowicz et al., 2020). An increased level of CD44 is associated with ccRCC. Two fluorophores PerCP and FITC-A were utilized to measure the fluorescence. The patient sample depicted fluorescence for the mix 2 antibody which indicates that the sample may indicate the presence of ccRCC. The 3 and 4 lab sessions demonstrated the ELISA experiments which were used to demonstrate the comparison between the concentration of IL-6 cytokine concentration between the healthy and patient samples. IL-6 is the cytokine that is associated with increased tumor proliferation and promotes metastases (Gudbrandsdottir et al., 2020). Increased concentration of the cytokine was visualized in the patient sample after plotting the graphs of OD and concentration which demonstrated the appearance of RCC. Overall all three laboratory procedures were ideal to attain ideal knowledge concerning the prognosis and detection of patients with ccRCC. The lab session was constructed to achieve considerable exposure to the diagnosis of ccRCC.

References

Alhajj, M., & Farhana, A. (2022). Enzyme-linked immunosorbent assay. In StatPearls [Internet]. StatPearls Publishing.

Bover, J., Ureña, P., Aguilar, A., Mazzaferro, S., Benito, S., López-Báez, V., ... & Cozzolino, M. (2018). Alkaline phosphatases in the complex chronic kidney disease-mineral and bone disorders. Calcified Tissue International, 103, 111-124. 10.1007/s00223-018-0399-z

DiGiuseppe, J. A., & Wood, B. L. (2019). Applications of flow cytometric immunophenotyping in the diagnosis and posttreatment monitoring of B and T lymphoblastic leukemia/lymphoma. Cytometry Part B: Clinical Cytometry, 96(4), 256-265. https://doi.org/10.1002/cyto.b.21833

Farber, N. J., Kim, C. J., Modi, P. K., Hon, J. D., Sadimin, E. T., & Singer, E. A. (2017). Renal cell carcinoma: the search for a reliable biomarker. Translational Cancer Research, 6(3), 620. 10.21037/tcr.2017.05.19

Fiedorowicz, M., Khan, M. I., Strzemecki, D., Orzeł, J., Wełniak-Kamińska, M., Sobiborowicz, A., ... & Czarnecka, A. M. (2020). Renal carcinoma CD105−/CD44− cells display stem-like properties in vitro and form aggressive tumors in vivo. Scientific Reports, 10(1), 5379. 10.1038/s41598-020-62205-6

Gudbrandsdottir, G., Aarstad, H. H., Bostad, L., Hjelle, K. M., Aarstad, H. J., Bruserud, Ø., ... & Beisland, C. (2021). Serum levels of the IL-6 family of cytokines predict prognosis in renal cell carcinoma (RCC). Cancer Immunology, Immunotherapy, 70, 19-30. 10.1007/s00262-020-02655-z

Herold, N. C., & Mitra, P. (2022). Immunophenotyping. In StatPearls [Internet]. StatPearls Publishing.

Hsieh, J. J., Purdue, M. P., Signoretti, S., Swanton, C., Albiges, L., Schmidinger, M., & Ficarra, V. (2017). Renal cell carcinoma. Nature Reviews Disease Primers, 3(1), 1-19. https://doi.org/10.1038/nrdp.2017.9

Landolt, L., Eikrem, Ø., Strauss, P., Scherer, A., Lovett, D. H., Beisland, C., & Marti, H. P. (2017). Clear cell renal cell carcinoma is linked to epithelial‐to‐mesenchymal transition and fibrosis. Physiological Reports, 5(11), e13305.10.14814/phy2.13305

Nabi, S., Kessler, E. R., Bernard, B., Flaig, T. W., & Lam, E. T. (2018). Renal cell carcinoma: a review of biology and pathophysiology. F1000Research, 7. 10.12688/f1000research.13179.1

Park, J., Shin, S. J., Shin, J., Lee, A. J., Lee, M., Lee, M. J., & Park, Y. (2023). Quantification of structural heterogeneity in H&E stained clear cell renal cell carcinoma using refractive index tomography. Biomedical Optics Express, 14(3), 1071-1081.https://doi.org/10.1364/BOE.484092

Schneider, M. R. (2021). Von Kossa and his staining technique. Histochemistry and Cell Biology, 156(6), 523-526.10.1007/s00418-021-02051-3

Signoretti, S., Flaifel, A., Chen, Y. B., & Reuter, V. E. (2018). Renal cell carcinoma in the era of precision medicine: from molecular pathology to tissue-based biomarkers. Journal of Clinical Oncology, 36(36), 3553. 10.1200/JCO.2018.79.2259

Tolmachev, V., Bodenko, V., Orlova, A., Schulga, A., Deyev, S. M., & Vorobyeva, A. (2023). Visualization of epithelial cell adhesion molecule‑expressing renal cell carcinoma xenografts using designed ankyrin repeat protein Ec1 labeled with 99m Tc and 125 I. Oncology Letters, 25(1), 1-10. https://doi.org/10.3892/ol.2022.13598

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