GFAT1: A Potential Prognostic Biomarker in Colorectal Cancer

@#Introduction: There is an increasing demand for additional techniques to diagnose and treat cancer including CRC or colorectal cancer effectively. Utilizing antibodies as biomarker could contribute to accurate diagnosis of cancer due to its high specificity and sensitivity. One of the etiologies of CRC progression was proposed as the alterations of hexosamine biosynthetic pathway which could subsequently influence the rate-limiting enzyme, glutamine-fructose-6-phosphate aminotransferase (GFAT1). These increased enzymatic activities resulted in an elevation of glucose uptake that provides nutrients facilitating the progression of cancer cells. Therefore, we attempted to determine the potential of GFAT1 as the biomarker for CRC by correlating its expression with clinicopathological features of the patients. Methods: A total of 132 10% formalin-fixed paraffin embedded tissue were retrieved. Immunohistochemistry (IHC) was performed on the tissue sections and digital images were subsequently acquired. All the images were automatedly analyzed using IHC Profiler. GFAT1 immunoreactivity in colorectal tissues was calculated using an adapted H-score formula. Clinicopathological features of the patients were statistically correlated with the status of GFAT1. Results: Colorectal adenocarcinoma tissues had the significantly highest GFAT1 H-scores with the mean of 103.18 compared to adenoma and non-tumor tissues. There have been no significant associations between clinicopathological characteristics of the patients and the status of GFAT1 except for tumor size. Conclusion: Immunoreactivity of GFAT1 was significantly different between non-tumorous tissues and adenocarcinoma as well as between adenoma and adenocarcinoma tissues. GFAT1 could serve as one of the prognostic biomarkers or useful targets.

Effect of different diluents on stability of mixed enzyme-labeled antibody / 中国生物制品学杂志

@#Objective To investigate the effect of different diluents on the stability of the mixed enzyme-labeled antibody,and screen a suitable diluent for the mixed enzyme-labeled antibody,which can protect the stability of the cocktail mixture of horseradish peroxidase(HRP) conjugated secondary antibody and alkaline phosphatase(AP) conjugated secondary antibody.Methods Using Tris-HCl buffer as the base solution,four different diluent formulations(A,B,C,D) were prepared with different kinds of stabilizers contained in each formula,such as protein,metalion,surfactant and bacteriostatic agent.Mixed enzyme-labeled antibodies were prepared with different diluents and stored at 2～8 ℃ and 37 ℃,which were detected for the stability by ELISA,enzyme activity assay and immunohistochemistry(IHC) staining.Results D solution [Tris(50 mmol/L)+BSA(1.5%,g/100 mL)+Tween-20(0.3%)+Proclin-950(0.1%,g/100 mL)+sodium chloride(150 mmol/L)+L-Ascorbic Acid(1 mmol/L)+L-methionine(5 mmol/L)+L-histidine(5 mmol/L)+sodium caseinate(1%,g/100 mL)+zinc chloride(0.1 mmol/L)+trehalose(8%,g/100 mL)] was of the optimal protective effect.When stored at 37 ℃ for 10 weeks,the working solution of HRP conjugated secondary antibodies and AP conjugated secondary antibodies showed the highest titer,the activity residual ratios of HRP and AP were 93.46% and96.07%,respectively,and there was no significant difference in IHC results.Conclusion This formula diluent can be used for the preparation of mixed enzyme-labeled antibody working solution.

HISTOMORPHOLOGICAL SPECTRUM OF UTERINE LEIOMYOMA VARIANTS – A RETROSPECTIVE STUDY IN TERTIARY CARE HOSPITAL

To study the histomorphological spectrum of uterine leiomyoma variants. This study is done over a period of three year (May 2019 to May 2022) in the Department of Pathology, LNMC, Bhopal. Total of 316 hysterectomy and 14 myomectomy specimens were studied. Specimens were fixed in formalin and paraffin embedded. H&E stained tissue sections were studied. In the study we performed retrospective analysis of hysterectomy and myomectomy specimen and 330 cases of leiomyoma were evaluated. Among 330 cases, 316(95.75%) were hysterectomy specimen for varying indication and 14(4.24%) were myomectomy specimen. Histologically the usual leiomyomas was comprising of 164(49.69%) cases followed by hyalinised leiomyoma 70(21.21%), myxoid leiomyoma 15(4.54%), hydropic change 12(3.63%), cellular 11(3.33%), lipoleiomyoma 10(3.03%), calcification 10(3.03%), infarct type necrosis 10(3.03%), mitotically active 8(2.42%), symplastic 7 (2.12%), schwanonian 6(1.81%), epithelioid 3(0.90%), dissecting leiomyoma 2 (0.60%) and stromal metaplasia (osseous and cartilaginous) 2(0.60%). Leiomyoma is the commonest benign smooth muscle tumor of the uterus with a number of histological variants. In this study conventional leiomyoma being the commonest variant followed by hyalinized leiomyoma, myxoid leiomyoma, hydropic leiomyoma and lipoleiomyoma. It is important to categorise various types of leiomyoma on histology to avoid misdiagnosis.

Alveolar rhabdomyosarcoma in children with histomorphological review.

Rhabdomyosarcomas (RMS) are aggressive malignant neoplasm of mesenchymal origin, related to skeletal muscle lineage. These are the most common soft tissue tumors in children. The diagnosis is made by microscopic analysis and ancillary techniques like immunohistochemistry, electron microscopy, cytogenetics and molecular biology. We encountered a case of a 03 years old child who presented with a tender, reddish, soft swelling over cheek for three weeks. The FNAC was reported as a small round cell tumor, Probably Primitive Neuroectodermal Tumor (PNET). The biopsy of tumor revealed a small round cell tumor with an alveolar pattern. Tumor giant cells were absent and mitotic figures were infrequent. Hence, differentials of alveolar rhabdomyosarcoma and PNET were rendered. Immunohistochemistry (IHC) demonstrated desmin positivity. Thus, a final diagnosis of alveolar rhabdomyosarcoma was offered.

Detección inmunohistoquímica del receptor del factor de crecimiento epidérmico: revisión de algunos aspectos técnicos relacionados / Immunohistochemical detection of the epidermal growth factor receptor: review of some related technical aspects

Introducción: En los últimos años la inmunohistoquímica (IHQ) se ha convertido en el método más usado en la determinación de la expresión del receptor del factor de crecimiento epidérmico (REGF). La falta de control de algunos aspectos técnicos durante su determinación en muestras de tejidos fijados en formol e incluidos en parafina, como por ejemplo, la fijación tisular y el procesamiento de las muestras, la elección de un método adecuado de reanimación antigénica, el empleo de diferentes anticuerpos anti-REGF y sistemas de detección, a llegado a tal punto que la confiabilidad y reproducibilidad de la detección inmunohistoquímica de la sobreexpresión del REGF está siendo fuertemente cuestionada. Objetivo: La estandarización de estos procedimientos constituye una de las metas más perseguidas actualmente, con el objetivo de continuar empleando la sobreexpresión del REGF por IHQ como criterio de elección y predictor de la respuesta al tratamiento, sin necesidad de recurrir a técnicas más complejas para su detección, disminuyendo en gran medida a la variabilidad intra e interlaboratorios. Métodos: Revisión y determinación de la pertinencia en el control de la técnica investigativa y viabilidad de la misma. Conclusiones: La determinación de la sobreexpresión del REGF por IHQ continúa siendo el método más empleado actualmente como predictor de la respuesta a la terapia contra el receptor. La heterogeneidad de las muestras, la falta de estandarización de los procedimientos inmunohistoquímicos empleados, la existencia de numerosos protocolos con un mismo fin, así como otras fuentes de variabilidad, han conducido a la obtención de resultados poco confiables y reproducibles

Background: In recent years, immunohistochemistry (IHC) has become the most widely used method in determining the expression of epidermal growth factor receptor (EGFR). The lack of control of some technical aspects during their determination in formaldehyde fixed and paraffin embedded tissue such as tissue fixation and sample processing, the choice of a suitable antigen retrieval method, the use of different anti-EGFR antibodies and the detection systems have come to the point that the reliability and reproducibility of the imunohistochemical analysis of EGFR overexpression is being seriously questioned. Objective: The standardization of these procedures is one of most pursued goals at present, with the aim to continue using EGFR overexpression by IHC as selection criteria and predictor of treatment response, without resorting to most complex techniques for its detection, greatly diminishing the intra and inter laboratory variation. Methods: A review and determination of the relevance in the control of the investigative technique and its feasibility was made. Conclusions: The determination of EGFR overexpression by IHC continues being the most used method at present as predictor of the response to the therapy against the receptor. The heterogeneity of the samples, the lack of standardization of the most used immunohistochemical procedures, the existence of several protocols with the same goal, as well as other sources of variability have led to obtaining hardly reliable and reproducible results

Comparative Analysis of the Distribution of Various Subtypes of Lymphoid Malignancies in Uttarakhand with other Regions in India.

Lymphoid malignancies (LM) form an umbrella term comprising both Hodgkin Disease (HD) and Non Hodgkin Lymphoma (NHL). A number of studies conducted in India and worldwide suggests that the disease exhibits similar pattern with contrasting regional variations. Examining regional variations is important as it may provide an insight to the etiological factor and pathogenesis of the disease. Aim: The aim of our study was to investigate the current pattern of lymphoid malignancies both HD and NHL in Uttarakhand and subsequently compare the results with other regions. Materials and Methods: We analyzed 116 cases of Lymphoid Malignancies that were reviewed over a period of 18 months. Both HD and NHL were diagnosed morphologically and then Immunohistochemistry (IHC) using CD3, CD15, CD20, CD30, and CD45 was employed to further subtype disease according to current WHO classification. Results: The lymphoid malignancies were further subdivided into HD and NHL. Nodular Sclerosis (NS) was the dominant subtype of HD in Uttarakhand (48.78%) and was comparable with results from other regions. Statistical analysis regarding distribution of various subtypes of HD in Uttarakhand and its comparison with three distinct geographical regions also showed p value < 0.241832 which was not statistically significant. However, amongst NHL Diffuse Large B cell Lymphoma (DLBCL) (54.66%) was the commonest subtype. Besides, a significant number of cases of Anaplastic Large Cell Lymphoma (12%) were also observed. Furthermore, statistical analysis showed that the distribution of various subtypes of NHL in Uttarakhand when compared to three distinct geographical regions was statistically significant (P value < 0.002808). Conclusion: Geographic differences in the incidence and histologic subtypes of Lymphomas do exist.

Evaluation of HER-2/neu status in breast cancer specimens using immunohistochemistry (IHC) & fluorescence in-situ hybridization (FISH) assay.

Background & objectives: Fluorescence in situ hybridization (FISH) is increasingly being recognized as the most accurate and predictive test for HER2/neu gene amplification and response to therapy in breast cancer. In the present study we investigated HER-2/neu gene amplification by FISH in breast carcinoma tissue specimens and compared the results with that of immunohistochemical (IHC) analysis. Methods: A total of 90 breast carcinoma tissue samples were used for immunohistochemical (IHC) and FISH analysis. IHC was performed by using mouse monoclonal antibody to the intracellular domain of HER-2/neu protein. Each slide was scored in a blinded fashion by two pathologists according to the manufacturer's recommended criteria. FISH analysis was performed on paraffin embedded breast tumour tissue sections. The polysomy for centromere 17 (Spec green signal) was read as green signals less than 4 as moderate polysomy, and more than 4 as highly polysomy. Results: Thirty of the 90 patients had negative results by IHC and FISH. Of the 28 patients with the score of 2+ by IHC, 20 were FISH positive for HER-2/neu gene amplification, three were FISH negative and five patients showed equivocal (1.8-2.2) results by FISH. These five cases were retested for IHC and FISH on different paraffin embedded tissue blocks, and all five were found positive for HER-2/neu gene amplification. Twenty five patients with the score of 3+ by IHC were FISH positive for HER-2/neu gene amplification (>2.2). Seven cases with the score of 3+ by IHC were FISH negative for HER-2/neu gene amplification (>2.2), and showed polysomy of chromosome number 17 high polysomy > 4. Interpretation & conclusions: Our results indicated that HER-2/neu status by FISH should be performed in all cases of breast tumour with a 2+ score by IHC. Cases demonstrating a 3+ score by IHC may be subjected to FISH to rule out polysomy of chromosome 17 which could be falsely interpreted as HER-2/neu overexpression by IHC analysis. There is also a need for establishing a clinically validated cut-off value for HER-2/neu FISH amplification against IHC which may be further compared and calibrated.

Assessment of HER-2/neu status in breast cancer using fluorescence in situ hybridization & immunohistochemistry: Experience of a tertiary cancer referral centre in India.

Background & objective: Determination of HER2 status in breast cancer has become important to identify potential candidates for anti-HER2 therapy. In this study we compared fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) for the determination of HER2 status in breast cancer patients referred to a tertiary care referral centre. Methods: A total of 200 cases of invasive breast cancer were evaluated for HER2 status using IHC and FISH and results were compared. Results: The IHC 3+ (93.9%) and IHC negative (85.9%) cases showed good concordance with the corresponding FISH results; while 66.6 per cent of IHC 2+ cases showed gene amplification by FISH. In addition, hormone receptor expression and HER2 gene status showed a statistically significant inverse association (P<0.05). Interpretation & conclusion: These findings reaffirm IHC as a prudent first-step to screen tissue samples for HER2 status and to determine suitability for technically demanding FISH test and the dual coloured FISH as a gold standard for determination of HER2/neu status in IHC equivocal cases of breast carcinoma.

EXPRESSION OF MATRIX METALLOPROTEINASE-9 IN HUMAN ABDOMINAL AORTIC ANEURYSMAL TISSUES / 药物分析学报

Objective To study the effects of MMP-9 (Matrix Metalloproteinase-9, MMP-9) in the pathogenesis of abdominal aortic aneurysms (AAAs) by localizing the expression of MMP-9 in the aneurysmal tissues. Methods By means of immunohistochemistry, the frozen sections (5 μm) with aneurysmal tissues (n = 10) were incubated with MMP-9 antibody-added agents, then the sections were stained and observed under the microscope to localize the expression of MMP-9, which displayed a brown precipitate within the arterial walls. The normal arterial wall tissues(n= 10)and the diseased arterial wall tissues from the arterial occlusive diseases (AODs) (n= 15) were also immunized exactly the same way as control. Results A quantity of positive granules which appeared within the aortic media showed the strong expression of MMP-9 in the AAAs, with the positive rate reaching 95%(19/20), while no expression of MMP-9 was observed in the normal artery. However, the scattered distributed positive granules were scen within the arterial wall of some cases of the AODs, implying the weak positive expression of MMP-9 in this disease with the positive rate of 26.7%(4/15). There was a significant difference of the expression of MMP-9 within the arterial wall between the AAAs and AODs(P＜0. 01). Conclusion High expression of MMP-9 within the aortic media faciliatates the degradation of collagen and elastin fibres and subsequent dilation of the aortic artery , thus playing an important role in the pathogenesis of AAAs. To refrain MMP-9 from enhanced expressing within the aortic wall is of clinical significance in the prevention and treatment of AAAs.

A Study for Detection of HBV by Sandwitch in situ Hybridization in Chronic Liver Disease / 대한내과학회지

OBJECTIVES: In situ hybridization(ISH) is widely applied as an effective method to detect the positive signal in the preservation of histological architecture with high specificity. The purpose of this study is to evaulate the clinical availability of SISH(sand-witch in situ hybridization) using HBsAg mRNA probe in biopsy specimen of patients with chronic liver disease. METHODS: SISH was employed to detect the HBsAg mRNA & DNA in 127 cases of chronic liver diseases and the results were compared with those obtained by immunohistochemistry and enzyme immunoassay methods, RESULTS: The HBsAg mRNA & DNA by SISH and the HBsAg by immmunohistochemistry were detected in 94 cases(92.2%) and 86 cases(84.3%) of 102 HBsAg seropositive cases, respectively. The detection of HBsAg mRNA & DNA by SISH was identified in 8 cases of 25 HBsAg seronegative cases of chronic liver disease, and the HBsAg by immunohistochemistry in 1 case. The detection rates of HBsAg mRNA & DNA by SISH were 78.3% in chronic persistent hepatitis, 81.7% in chronic active hepatitis, 87.5% in liver cirrhosis, and 70.6% in hepatocellular carcinoma. The positive rates of HRsAg by immunohistochemistry were 56.5% in chronic persistent hepatitis, 71.8% in chronic active hepatitis, 87.5% in liver cirrhosis, and 52.9% in hepatocellular carcinoma. The detection of HBsAg mRNA & DNA by SISH is more sensitive than that of HBsAg by immunohistochemistry. CONCLUSION: SISH using HBsAg mHNA probe is a useful and sensitive method to detect HBV in chronic liver disease, and it is more sensitive than immunohistochemistry.