Evaluation of Plasma Glycosaminoglycan in monitoring the patients of Renal cell carcinoma after Nephrectomy

Renal cell carcinoma is the thirteenth most widespread malignancy worldwide and is the leading cause of urogenital tract cancers. Worldwide, renal cell carcinoma is sixth most prevalent cancer in males and tenth most in females. Its diagnosis is based on histological findings and CT scans that were performed for isolated symptoms. Treatment options include chemotherapy, radiofrequency ablation, nephron-sparing surgery, and radical nephrectomy. Therefore, there is a need for an early non-invasive bio marker for diagnosis, disease surveillance, as well as for monitoring of RCC after therapy. Objective: To determine the role of plasma glycosaminoglycans levels for monitoring and prognosis of renal cell carcinoma patients after nephrectomy. Methods: A Cross sectional study was taken place at Armed forces institute of Pathology in collaboration with Armed forces institute of urology from July 2021 to Dec 2022. A total of 124 samples were included in study, out of which 62 samples were collected from pre-operated patients and another 62 from same RCC patients after operation. Plasma Glycosaminoglycans levels were analyzed through manual enzyme linked immunosorbent assay by non-competitive sandwich technique. Quantitative variables were calculated by means and standard deviation while qualitative variables were assessed as frequency and percentage. Paired t test was applied to check statistical significance of the difference between the means of pre-operative and post-operative cases. Results : Mean age of total population was 39.24±15.90 years. Taking in account age, gender and staging of pre-and post-operative patients, the results revealed significant post-Surgical decrease in plasma


Introduction
Renal cell carcinoma (RCC) is the thirteenth most widespread malignancy worldwide and ranks third amongst the leading reasons of urogenital tract cancers [1].Renal cell carcinoma is responsible for almost 90% of all renal malignancies.Worldwide, RCC is sixteenth most prevalent cancer in males and tenth most in females.It primarily affects patients over the age of 55(median age 64 years) [2].
Although there is lack of reliable information about the incidence of renal cell carcinoma in Pakistan, a study reported 1.8 percent prevalence of RCC among all tumors in males in a sample of 2393 cases performed at Agha Khan University in Karachi [3].During the last decade, there has been a spike in RCC all over the world.
One-third to one-fourth of patients have metastatic cancer at the time of diagnosis, while bilateral tumors are seen in just 2% of cases [4].
Factors leading to increased risk for renal cell carcinoma are male sex, adiposity, old age, alcoholism, persistent hemodialysis, high blood pressure, polycystic kidney disease, and various genetic disorders.Initial clinical appearances of RCC are different and may bring about an assortment of vague and often misattributed side effects.Just 10% of RCC patients come with typical flank mass, discomfort, and hematuria and these individuals frequently have progressed illness.Over 40% of RCC patients have no such symptoms [4].
The best way to detect renal masses is with a triple-phase contrast CT scan.These masses are frequently identified during an abdominal or chest CT scan that was initially performed for isolated symptoms.
necessitates more testing, such as imaging, to arrive at a diagnosis and treatment plan The study will help to establish a noninvasive test for monitoring of post-surgical patients which will be beneficial for both patients and the treating surgeons.A standardized and pre-tested questionnaire was used to estimate socioeconomic and demographic variables.

Results
Total sample size was 124 out of which 62 samples were collected from pre-operated patients and another 62 from same RCC patients after operation.Healthy individuals (n= 120) were included as controls.Mean age of the total population (n=244) was operative and post-operative patients as shown in table 1.

Reference Interval
Reference interval computed based on mean and ±2SD because data was parametric and reference interval computed on 120 participants who were disease free.

Reference intervals of
Glycosaminoglycans (GAG) levels of local adult population is 11.67-37.7mg/l in males and 11.71-32.73mg/l in females.These GAG adjustments may start from the tumor and that the liver keeps up plasma GAG homeostasis regardless of changes in the tumor trouble [17].Nonetheless, this theory matches with the perception that GAG scores appear to diminish postoperatively, which may be adequate to catch variances after surgery.

Inferential Statistics
In past investigations, GAG scores

Conclusion
The

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. Active surveillance, chemotherapy, radiofrequency ablation, nephron-sparing surgery (NSS) and radical nephrectomy are all options for treatment.NSS or partial nephrectomy is the preferred therapeutic option for minor isolated renal tumors as compared to radical nephrectomy.NSS provides the benefit of retaining renal function while also lowering cardiovascular mortality [6].Authoritative diagnosis is based on histological findings, but it is an intrusive procedure and may cause damage to kidneys.Therefore, there is a need for an early non-invasive bio marker for diagnosis, disease surveillance, as well as for monitoring of RCC after therapy.Plasma Glycosaminoglycan (GAG) levels is emerging as a promising marker for diagnosis and monitoring of surgically treated RCC patients.Glycosaminoglycan has viscous, lubricating characteristics similar to mucous secretions therefore they are also known as mucopolysaccharides [7].These are found in the extracellular matrix and on the surfaces of animal cells.They have been found to bind and modulate a variety of proteins including cytokines, chemokines, enzymes, growth factors (GF), morphogens and adhesion molecules [8].Glycosaminoglycans include far reaching capacities inside the body.They assume a critical part in the phone flagging cycle, including guidelines of cell development, multiplication, advancement of cell grip, anticoagulation, and wound repair [9].Urinary glycosaminoglycan excretion is enhanced in several disseminated cancers.In Renal cell carcinoma patients, GAG excretion in the urine is a reliable diagnostic marker for determining tumor size and unilocularity or multilocularity [10].Keeping in view, the importance of plasma GAG levels in the diagnosis and prognosis of RCC, our study aims to highlight the importance of GAG levels in post-surgical phase of RCC.This study will play a pivotal role in establishing plasma GAG levels as a significant marker for assessing the efficiency of nephrectomy in such patients.

For
estimation of GAG levels, 3ml blood sample was collected from study subjects from the antecubital vein and placed in an ethylenediaminetetraacetic acid (EDTA) tube.Then sample was sent to laboratory and centrifugation was done at 2000-3000 RPM for approximately 20 minutes within two hours of sample collection.Plasma GAG levels were analyzed through manual enzyme linked immunosorbent assay by non-competitive sandwich technique [Human GAGs (Glycosaminoglycan) ELISA Kit (G-Biosciences)].The principle was based on antigen antibody interaction.Human GAG antibody was pre-coated on the plate.GAG antigen existing in the sample was added and binds to antibody coated on the well.Result of the samples were analyzed using ELISA plate reader [specifications 96T (8 X 12) storage -20 0 C) Data was entered in the statistical software package (SPSS version 21).Quantitative variables were calculated by means and standard deviation while qualitative variables were computed as frequency and percentage.Kolmogrov-Smirnov test was applied to check normality of data.Paired ttest was applied for inferential statistics to check statistically significant difference in means among the groups.P-value of ≤ 0.05 was taken as significant.

NUSTTable 2 .
Journal of Natural Sciences, Vol. 8, Issue 2, 2023 Evaluation of Plasma Glycosaminoglycan in monitoring the patients of Renal cell carcinoma after Nephrectomy 6 Reference intervals of Glycosaminoglycans levels.

Figure 3 .
Figure 3. Gender wise distribution of renal cell carcinoma staging diminished to ordinary levels in eight patients with no disease, however these examples were gotten a very long time after the surgery.The high affectability could be valuable for applications significant in the clinical administration of RCC, for example, observation for repeat after surgery.It has been indicated that plasma GAGs can possibly make up for the absence of insignificantly obtrusive biomarkers in the administration of RCC [18].In spite of the away from of the GAG level as a biomarker of direct clinical use, this investigation ought to be additionally assessed to organize phase II and III of clinical trials.According to some researcher's noninvasive assays can be used to assess particles in serum.They identified altered serum protein production in RCC patients using immunoassays such as ELISA or immunonephelometry, as well as mass spectrometry.That give helpful indicative or potentially prognostic data.It is conceivable that distinctions in identity and way of life can prompt diverse plasma GAG standard levels in various populaces, thus wide associate examination is additionally proposed [19][20].Right now, there is no symptomatic biomarker that has entered routine practice for metastatic ccRCC.It would without a doubt address a significant clinical headway if changes in the GAG profile could establish a marker of the event of the Infection.This is a quickly developing and promising space of exploration for the plan and advancement of novel incredible specialists for finding and anticipation, drug conveyance and atomic focused on treatment in disease.The current study is limited to phase-I clinical trial for GAG levels in plasma but not in urine for the purpose of investigating its role in diagnosis of RCC that is proven to be effective by this study.The study has NUST Journal of Natural Sciences, Vol. 8, Issue 2, 2023 Evaluation of Plasma Glycosaminoglycan in monitoring the patients of Renal cell carcinoma after Nephrectomy 10 concise sample size.There is a need to conduct further multi-centric studies before establishing the role of GAG levels in postnephrectomy patients.More cohort study should be performed proceeding to phase-II and phase-III clinical trials.Further studies should be conducted which include serial monitoring of post-nephrectomy patients with GAG levels.Such studies will help to signify the role of GAG level as a new emerging biomarker in post treatment phase of RCC.

Table 1 :
Descriptive statistics of the study Mean age of males was 34.74±14.88yearsandmean age of females were 46.28±14.92years.Total males were 149 (61.1%) while females were 95(38.9%).asshown in figure1.In pre-operative and post-operative group males were 28 and female were 34.

Table 3 .
Glycosaminoglycan levels in Preoperative and Post-operative group

Table 4 :
Comparison of GAGs levels inPre-operative and Post-operative group