Archival Paraffin Blocks Research Articles

Different types of tumor microvessels in stage I-IIIA squamous cell lung cancer and their clinical significance.

Lung cancer (LC) is the leading cause of morbidity and mortality among malignant neoplasms. Improving the diagnosis and treatment of LC remains an urgent task of modern oncology. Previously, we established that in gastric, breast and cervical cancer, tumor microvessels (MVs) differ in morphology and have different prognostic significance. The connection between different types of tumor MVs and the progression of LC is not well understood. To evaluate the morphological features and clinical significance of tumor MVs in lung squamous cell carcinoma (LUSC). A single-center retrospective cohort study examined medical records and archival paraffin blocks of 62 and 180 patients with stage I-IIIA LUSC in the training and main cohorts, respectively. All patients underwent radical surgery (R0) at the Orenburg Regional Cancer Clinic from May/20/2009 to December/14/2021. Tumor sections were routinely processed, and routine Mayer's hematoxylin and eosin staining and immunohistochemical staining for cluster of differentiation 34 (CD34), podoplanin, Snail and hypoxia-inducible factor-1 alpha were performed. The morphological features of different types of tumor MVs, tumor parenchyma and stroma were studied according to clinicopathological characteristics and LUSC prognosis. Statistical analysis was performed using Statistica 10.0 software. Univariate and multivariate logistic regression analyses were performed to identify potential risk factors for LUSC metastasis to regional lymph nodes (RLNs) and disease recurrence. Receiver operating characteristic curves were constructed to discriminate between patients with and without metastases in RLNs and those with and without disease recurrence. The effectiveness of the predictive models was assessed by the area under the curve. Survival was analyzed using the Kaplan-Meier method. The log-rank test was used to compare survival curves between patient subgroups. A value of P < 0.05 was considered to indicate statistical significance. Depending on the morphology, we classified tumor vessels into the following types: normal MVs, dilated capillaries (DCs), atypical DCs, DCs with weak expression of CD34, "contact-type" DCs, structures with partial endothelial linings, capillaries in the tumor solid component and lymphatic vessels in lymphoid and polymorphocellular infiltrates. We also evaluated the presence of loose, fine fibrous connective tissue (LFFCT) and retraction clefts in the tumor stroma, tumor spread into the alveolar air spaces (AASs) and fragmentation of the tumor solid component. According to multivariate analysis, the independent predictors of LUSC metastasis in RLNs were central tumor location (P < 0.00001), the presence of retraction clefts (P = 0.003), capillaries in the tumor solid component (P = 0.023) and fragmentation in the tumor solid component (P = 0.009), whereas the independent predictors of LUSC recurrence were tumor grade 3 (G3) (P = 0.001), stage N2 (P = 0.016), the presence of LFFCT in the tumor stroma (P < 0.00001), fragmentation of the tumor solid component (P = 0.0001), and the absence of tumor spread through the AASs (P = 0.0083). The results obtained confirm the correctness of our previously proposed classification of different types of tumor vessels and may contribute to improving the diagnosis and treatment of LUSC.

Data retrieval from archival renal biopsies using nonlinear microscopy.

Thorough examination of renal biopsies may improve understanding of renal disease. Imaging of renal biopsies with fluorescence nonlinear microscopy (NLM) and optical clearing enables three-dimensional (3D) visualization of pathology without microtome sectioning. Archival renal paraffin blocks from 12 patients were deparaffinized and stained with Hoechst and Eosin for fluorescent nuclear and cytoplasmic/stromal contrast, then optically cleared using benzyl alcohol benzyl benzoate (BABB). NLM images of entire biopsy fragments (thickness range 88-660 μm) were acquired using NLM with fluorescent signals mapped to an H&E color scale. Cysts, glomeruli, exudative lesions, and Kimmelstiel-Wilson nodules were segmented in 3D and their volumes, diameters, and percent composition could be obtained. The glomerular count on 3D NLM volumes was high indicating that archival blocks could be a vast tissue resource to enable larger-scale retrospective studies. Rapid optical clearing and NLM imaging enables more thorough biopsy examination and is a promising technique for analysis of archival paraffin blocks.

The evaluation of isocitrate dehydrogenase 1 immunohistochemical expression and methylation status of O6-methylguanine-DNA-methyltransferase in glioblastoma patients and their impact on survival rates

Introduction Glioblastoma (GBM) is a highly aggressive lethal glial tumor with a dismal prognosis. The success of different therapeutic approaches depends mainly on genetic heterogeneity. Mutation in the isocitrate dehydrogenase (IDH) 1 gene and hypermethylation of O6-methylguanine-DNA-methyltransferase (MGMT) are potential biomarkers for predicting GBM prognosis and response to treatment. Aim This study aimed to investigate IDH1 immunohistochemical expression and MGMT promoter methylation status in a subset of Egyptian patients diagnosed with GBM and their correlation with overall survival (OS) and progression-free survival (PFS). Methods This study was conducted on 30 archived paraffin blocks of GBM patients with available clinical and follow-up data. The patient’s medical files were revised to document their clinical data, and immunostaining was done for detecting IDH1 expression. Polymerase chain reaction (PCR) was performed to assess MGMT promoter methylation. Kaplan-Meier estimates and log-rank tests were used to assess OS and PFS. Results Out of the 30 GBM cases, 46.7% showed positive IDH1 immunostaining, and 63.3% were MGMT-methylated. Positive IDH1 immunostaining and methylated MGMT cases were significantly associated with better OS and PFS, 6- and 12-month follow-up postchemotherapy (P&lt;0.05). In contrast, the extent of surgery was only associated with PFS (P&lt;0.05), but not with OS (P&gt;0.05). Conclusion This study highlights the value of investigating IDH1 mutation and MGMT promoter methylation as potential biomarkers for predicting the prognosis of patients with GBM and the possibility of developing future targeted therapy. However, further studies on larger scales are needed to validate these findings.

Topoisomerase IIalpha (TopoIIa) A New Promising Marker for Early Detection of Laryngeal Squamous Cell Carcinoma: An Immunohistochemical Study.

the aim of this study is evaluation of Topo IIa expression in laryngeal squamous cell carcinomas and correlation of this expression with various clinicopathological parameters. ninety cases of laryngeal squamous cell carcinoma archived paraffin blocks were collected in the form of total laryngectomies. Each paraffin block was re-cut by rotatory microtome at 4 μm thickness and stained by hematoxylin and eosin for routine histopathological examination and on charged slides for immunohistochemistry using an automated staining system with antibodies against Topo IIa. Mainly nuclear and slightly cytoplasmic staining was considered positive. Percentage of positive Topo IIa cells was graded then grouped into low expression and overexpression. Topo IIa overexpression was seen in 91.1% of cases, while low expression was noticed in the remaining 8.9% of cases. There were statistically significant correlations between Topo IIa expression and tumor histological grade, lymph node metastasis and T stage as well as statistically significant positive correlation between Topo IIa expression as we progress from normal to dysplastic/in situ up to malignant transformation. High expression of Topo IIa may indicate more aggressive tumor and may play a role in tumorogenesis in laryngeal squamous cell carcinoma.

Standardization of Grocott's methenamine (hexamine) silver method for glycogen demonstration in liver tissue.

Demonstration of glycogen can be done in different lesions and is considered diagnostically significant, mainly in some tumors. Glycogen staining is affected by the type of fixative, the temperature of fixation, and the staining technique.Grocott's methenamine (hexamine) silver technique quality was assessed after four different types of fixatives at two different temperatures [Bouin's solution, 10% neutral buffered formalin (NBF), 80% alcohol, and Rossman's solution at room temperature (RT) and 4°C, for 24h]. These variables were studied to optimize this technique for glycogen demonstration. Archived paraffin blocks were used in this study. They were prepared from one rabbit's liver, and 32 paraffin sections were prepared and stained with Grocott's methenamine (hexamine) silver technique. Eightypercent alcohol provided the highest staining quality scores at both RT and 4°C in comparison with the other fixatives. We concluded that 80% alcohol at 4°C seems to be the fixative of choice for glycogen with the Grocott's methenamine (hexamine) silver technique at the level of this study.

Pre-Vaccination Human Papillomavirus Genotypes and HPV16 Variants among Women Aged 25 Years or Less with Cervical Cancer.

In 2007, Australia introduced a national human papillomavirus (HPV) vaccination program. In 2017, the onset of cervical screening changed from 18 to 25 years of age, utilising human papillomavirus (HPV) nucleic acid testing. The objective of the study is to describe the HPV genotypes and HPV16 variants in biopsies from women ≤ 25 years of age with cervical carcinoma (CC) (cases), compared with those aged >25 years (controls), in a pre-vaccination cohort. HPV genotyping of archival paraffin blocks (n = 96) was performed using the INNO-LiPA HPV Genotyping assay. HPV16-positive samples were analysed for variants by type-specific PCR spanning L1, E2 and E6 regions. HPV16 was the commonest genotype in cases (54.5%, 12/22) and controls (66.7%, 46/69) (p = 0.30), followed by HPV18 (36.3%, 8/22 vs. 17.3% 12/69, respectively) (p = 0.08). Furthermore, 90% (20/22) of cases and 84.1% (58/69) of controls were positive for HPV16 or 18 (p = 0.42); 100% (22/22) of cases and 95.7% (66/69) of controls had at least one genotype targeted by the nonavalent vaccine (p = 0.3). The majority of HPV16 variants (87.3%, 48/55) were of European lineage. The proportion of unique nucleotide substitutions was significantly higher in cases (83.3%, 10/12) compared with controls (34.1%, 15/44), (p < 0.003, χ2, OR 9.7, 95%CI 1.7-97.7). Virological factors may account for the differences in CCs observed in younger compared with older women. All CCs in young women in this study had preventable 9vHPV types, which is important messaging for health provider adherence to new cervical screening guidelines.

Prognostic value of androgen receptor expression in different molecular types of breast cancer in women.

Breast cancer is a common women's disease. Usually, oestrogen is blamed in the aetiology and correlated with the prognosis; however, androgens are recently raising concern about its role in the breast cancer treatment and prognosis. In this study we retrieved archival paraffin blocks of breast cancer patients and stained it for androgen. Thereafter, we compared clinico-epidemiologic parameters, histopathology, neoadjuvant response and recurrence rate and pattern among patients with and without androgen receptor (AR) expression. In total, 119 patients fulfilled enrolment criteria; AR expression were present in 77.3% of the patients. AR expression was associated with less grade III (6.8% versus 36.4%), and less triple negative (6.2% versus 25%), but similar overall recurrence rate (25% versus 22.2%). However, distant recurrence was significantly higher in androgen positive patients (91.3% versus 33.3% of all recurrences). Androgen expression appears to be common among breast cancer, but with no clear implication in tumour aggressiveness or effect on the rate of recurrence. However, being commonly associated with distant spread may have an impact on survival of the patients.

Expression of Anaplastic Lymphoma Kinase (ALK) in glioma and possible clinical correlations. A retrospective institutional study

BackgroundGlioblastoma is considered the most aggressive primary brain tumor. Recurrence after treatment is a significant problem with a failed response to optimal treatment. The recurrence of GBM is linked to different cellular and molecular pathways. Nationwide, in Egypt, astrocytic tumors are the most commonly diagnosed CNS tumor. Anaplastic Lymphoma Kinase (ALK CD246) is an enzymatic protein (RTK) belonging to the insulin receptors superfamily. MethodsThis is a retrospective study including sixty cases of astrocytic tumors (males = 40, mean age = 31.5), (females = 20, mean age = 37.77) obtained through collecting archived paraffin blocks of astrocytic tumor from the Pathology Department, Cairo University Faculty of Medicine during the period from January 2015 till January 2019. All cases were evaluated for ALK expression trying to find any clinical correlations with the clinical data. ResultsCorrelations were made using a scatterplot matrix correlogram. There was a significant correlation between tumor recurrence and ALK expression (r = 0.8, P < 0.01), and incidence of postoperative seizures (r = 0.8, P < 0.05), and between mean age and score tumor (r = 0.8, P < 0.05). ConclusionExpression of ALK was found to be abundant among high-grade gliomas and tumor recurrence rate was higher in ALK-positive patients. Further studies are needed to evaluate the potential use of ALK as a prognostic marker in cases of GBM.

Immunohistochemical Expression of PD-L1 and IDH1 with Detection of MGMT Promoter Methylation in Astrocytoma

Programmed death ligand 1 (PD-L1) expression was suggested as a poor prognostic predictor for glioblastoma. While isocitrate dehydrogenase (IDH) has been linked to enhanced overall survival in glioma cells. In glioblastoma patients receiving treatment with alkylating drugs, the methylguanine-DNA methyltransferase (MGMT) promoter's methylation status has been discovered as a potent and distinct predictor of good survival. In this study, we aimed to investigate the expression rate of PD-L1, IDH1, and MGMT methylation in patients with different grades of astrocytoma. The present retrospective study retrieved the data and archived paraffin blocks of 60 cases of astrocytoma. Immunohistochemical evaluation was done to assess the expressions of PD-L1 and IDH1, Methylation-specific-PCR was used to investigate the MGMT promoter. This study included astrocytoma grade II 18% (11/60), grade III 22% (13/60), grade IV 60% (36 cases). PD-L1 expression was detected in 82% of all studied cases (49/60) while IDH1 mutant astrocytoma were 73% (44/60) & methylation was reported in 58.3% (35 cases). High grade astrocytoma showed highrer expression of PD-L1 & IDH1 but with insignificant correlation (p=0.989). There is a relatively high expression of PD-L1 and IDH1 in patients with astrocytoma. More than half of the patients presented with MGMT promoter methylation. Further studies with larger sample size are required to investigate the association between these biomarkers and characteristics of patients with astrocytoma.

Expression and Clinical Significance of Thyroid-stimulating hormone receptor in the Subtypes of Papillary thyroid carcinomas.

To investigate the expression of TSH receptors (TSHR) in various subtypes of Papillary thyroid carcinomas (PTC) by immunohistochemistry. Retrospective analyses were carried out to the clinical data of 108 PTC patients randomly admitted into the Department of Thyroidthyroid surgery thyroid surgery and Breast Surgery, The Second Hospital of Hebei Medical University from March 2020 to December 2020. The archived paraffin blocks of the 108 cases as well as 18 contiguous normal thyroid tissues (control group) were taken from the Department of Pathology of The Second Hospital of Hebei Medical University. The pathological types of all PTC tissues were detected and the expression of TSHR was determined. TSHR expression was 86.11% positive in PTC tissues; with 85.00% positive in classical group; with 75.86% positive in micro group; with 84.61% positive in follicular group; with 83.33% positive in oncocytic group; with 50.00% positive in invasive group. TSHR expression was 100% in normal thyroid tissues. So TSHR expression in normal thyroid tissues is significantly higher than that in PTC; TSHR expression in microcarcinoma is stronger than in the other subtypes; there is no significant difference among the other subtypes. TSH suppression works better on microcarcinoma than on the other subtypes. And the effects on non-invasive subtypes are better than on invasive subtypes.

Immunohistochemical Study of Cancer Stem Cell marker, Tight Junction Protein, and Lymphatic Density in Malignant Salivary Gland Tumors

Background: CD117/c-kit, is a powerful stem cell marker for malignant salivary gland tumors in which dysregulation of c-kit is closely associated with impairment of cell adhesion molecules and cancer metastasis.&#x0D; Objective: The main purpose of this work is to evaluate the immunohistochemical expression of c-kit, and claudin-1 and measure the density of lymph vessels (LVD) in common malignant salivary gland tumors by using podoplanin (D2-40) antibody.&#x0D; Materials and Methods: Immunohistochemical staining with streptavidin peroxidase was used to analysis the expression of c-kit, claudin-1 and stained podoplanin (D2-40) lymphatic vessels on fifty archival paraffin blocks of malignant salivary gland tumor (MSGTs) cases included 20 cases of AdCC, 11 cases of MEC, 10 cases of CXPA, 6 cases of AcCC, and 3 cases of PAC.&#x0D; Results: The immunopositivity of c-kit (CD117) was detected in 44/50 (88%) of studied cases, whereas, claudin-1 protein was observed in 35 (70%) of our specimens of MSGTs. Count down of stained lymph vessels between examined cases was, MEC on the top, followed by CXPA, AdCC, PAC and AcCC. A direct correlation was observed between c-kit and lymphatic density, on the other hand, the inverse correlation was found d between c-kit and cld-1, as well as, between cld-1 and lymphatic density&#x0D; Conclusion: Up regulation of cancer stem cell marker c-kit (CD117) expression is associated with decrease of tight junction protein cld-1 and increase the density of stained lymphatic vessels by podoplanin (D2-40) antibody which confirms the using of c-kit inhibitor to improve treatment strategy of malignant salivary gland tumors.

MOLECULAR GENETIC CHANGES IN THE TESTIS TISSUES OF COVID-19 PATIENTS

Despite some progress in studying the impact of COVID-19 infection on the human body, many issues related to emerging pathological processes after the transfer of the disease, as well as the development of the so-called «post-COVID» syndrome, remain unresolved. One such issue is the impact of SARS-CoV-2 on male fertility. The results of previous studies in this direction are contradictory, and therefore at the moment there is no clear evidence of direct damage to male gonads by coronavirus. Thus, the hypothesis of potential testicular targeting for SARS-CoV-2 needs to be confirmed. The aim of the study was to assess the molecular genetic profile of samples of testicular tissue preparations from patients with COVID-19. Testicular tissue samples from patients with confirmed COVID-19 (n=96, age 25-91 years) were studied by real-time polymerase chain reaction to determine the expression of SARS-CoV-2 viral RNA and genes encoding protein complexes of ACE- 2 and Furin. The exclusion criteria were: mumps, infertility, sepsis, bacterial infection, carriage of HIV, hepatitis B and C, Epstein-Barr. The control of observations (n=20) consisted of archival paraffin blocks of autopsy material of normal testicles, obtained no later than 6 hours after the declaration of biological death, without macroscopic signs of the presence of an inflammatory and / or tumor process, all patients of this subgroup were fertile and had not previously been exposed to toxins or drugs. As a result of the study, in patients affected by COVID-19, the presence of the genetic material of the coronavirus in the testicles was recorded. In addition, an increased expression of ACE-2 and Furin was found in the testicular tissue, which determines favorable conditions for SARS-CoV-2 damage. Thus, based on the results of PCR testing of testicular tissue preparations for the presence of SARS-CoV-2 virus RNA, assessment of the expression of ACE-2 and Furin, it is possible to assert with a high probability the potential targeting him on male germ cells, Sertoli and Leydig cells.

Assessment of myeloperoxidase (Mpo) gene polymorphism in cervical cancer

Background/Aim: Cervical cancer (CC) is the most common gynecological malignancy in women. In spite of a variety of treatment protocols, it is necessary to carefully investigate all factors that play a role in the pathogenesis of these tumors which may have mortal progression. In this context, in our study we aimed to assess the myeloperoxidase (MPO) gene polymorphism, an important inflammatory enzyme, among cervix cancer cases. Methods: In this cross-sectional study, 79 cases diagnosed as cervical carcinoma between 1992-2012 is included. The cases without archival paraffin blocks and clinical follow-ups are excluded. All slides with tumor involvement are reviewed and the ones which demonstrate tumor’s characteristics are determined. After block determination 3 sections with 10-micron thickness obtained from paraffin blocks and MPO gene polymorphism was shown using acil restriction endonuclease enzyme with the restriction fragment length polymorphism (RFLP) method after polymerase chain reaction (PCR). The histopathological parameters including tumor stage and type, lymph node metastasis, ovary and endometrium involvement, recurrence and late metastasis are compared with genotype using chi-square and Fisher’s exact test. Results: The mean age of cases was 51.3 (10.9) years. Of 79 cases, 29 (36.7%) had AG (adenine-guanine) and 50 (633%) had GG (guanine-guanine) genotypes. Only endometrium involvement was identified to have a statistically difference with MPO gene polymorphism among the assessed histopathologic parameters (P=0.015). When clinical parameters are assessed, there was no difference identified between genotype and mortality (P=0.622). Conclusion: Cervical cancer is thought to have progressive and regressive characteristics of tumor development due to the inflammatory response of the host. Within this framework, in our study assessing gene polymorphism of one of the inflammatory response foundation stones of MPO, we identified more endometrial involvement for cases with AG genotype. We believe this significance will be encountered for more parameters in broad case series.

VEGF-R1/Flt-1 Receptor as a Characteristic of the Angiogenic Phenotype of Uveal Melanoma

Thanks to new fundamental data, our understanding of the angiogenesis’ role and its molecular participants has changed. Molecular genetic mechanisms of activation of signaling pathways of proliferation, malignancy, suppression of the tumor cells’ apoptosis, carried out through the interaction of the VEGF molecule with its receptors, have been deciphered. Molecular genetic mechanisms of activation of signaling pathways of proliferation, malignancy, suppression of apoptosis of tumor cells, carried out through the interaction of the VEGF molecule with its receptors, have been deciphered. The doctrine of angiogenesis has changed. Angiogenesis began to be considered in the aspect of the increasing anaplasia mechanism, accelerating proliferation, the formation of a clone of stem tumor cells, highly resistant to chemotherapy and radiation therapy with a high potential for metastasis. It is time to reconsider the role of individual biological markers of angiogenesis in their suitability in predicting disease outcome and evaluating them as a potential target for targeted therapy. In this aspect, uveal melanoma (UM) as a model of an extremely aggressive malignant tumor using its angiogenic phenotype to accelerate hematogenous metastasis is of particular interest. One of the characteristics of the angiogenic phenotype is VEGF-R1/Flt-1. The purpose: to study the expression of the VEGF-R1/Flt-1 receptor as a characteristic of the angiogenic phenotype of UM in correlation with its clinical and morphological indicators and the outcome of the disease. The authors conducted a retrospective study on 98 archival paraffin blocks of the eyes of patients with UM. The following general patterns of Flt expression in UM cells are revealed: Basic expression takes place in the smallest tumor proliferates UM (T1). Moreover, overexpression (IGH-gradation III) of Flt in the nucleus (39.7 %) and cytoplasm (36.3 %) occurred in every third patient with UM in stage T1. The peak of reception for the maximum average percentage of positive cells and the average cellular IGH gradation of expression was recorded at the T2 stage, after which there was a slow decline to the T4 stage. The number of overexpressive Flt in the nucleus and cytoplasm of UM cells increased x 2 times (75.3 %) to the T2 stage. The peak of Flt nuclear expression in terms of the number of immunopositive cells was also recorded at the T2 stage. The authors concluded that VEGF-R1/Flt-1 expression is a very important characteristic of the UM angiogenic phenotype. In the vast majority of UM, there is an expression of the VEGF-R1/Flt-1 receptor in the nucleus and cytoplasm of tumor cells. The revealed correlations of VEGF-R1/Flt-1 expression with the volume and histological type of tumor, disease stage and metastasis allow them to consider Flt-1 an important indicator associated with the pathogenesis and prognosis of uveal melanoma and a potential target for targeted therapy. A prognostic adverse factor in the aspect of the prognosis of the risk of metastases should be considered the index of the ratio Flt C/Flt N ≥ 3.

Expression of GATA3 and Cytokeratin 14 in Urinary Bladder Carcinoma (Histopathological and Immunohistochemical Study)

BACKGROUND: Urothelial carcinoma (UC) with squamous differentiation (SD) is the most common histologic variant of bladder carcinoma and its presence is associated with poor prognosis which may need early radical cystectomy to avoid progression and recurrence. It is difficult to detect few foci of SD, especially nonkeratinizing or early switch from urothelial to squamous epithelium on only morphological basis. Combination of GATA3 and Cytokeratin 14 (CK14) could be helpful in differentiating pure UC, UC with SD and pure squamous cell carcinoma (SCC). AIM: Assessment of GATA3 and CK14 expression in urinary bladder carcinoma and correlation with clinical and histopathological variables, for both diagnostic and prognostic purposes. MATERIALS AND METHODS: Sixty cases of archived paraffin blocks of urinary bladder carcinoma were tested for GATA3 and CK14 expression by immunohistochemistry using a rabbit monoclonal antibody against human CK 14 and mouse monoclonal antibody against GATA3, respectively. RESULTS: There is a significant correlation between GATA3 immunohistochemical expression and histological tumor subtypes of bladder carcinoma (p &lt; 0.001), i.e. the GATA3 is a useful marker for urothelial origin especially in papillary UC. There is a significant correlation between GATA3 immunohistochemical expression and UC grade (p &lt; 0.001). CK14 showed positive cytoplasmic staining in 9/14 (64.3%) cases of UC with SD and (13/13) (100%) cases of pure SCC and negative in 33/33(100%) cases of UC other than UC with SD. CK14 had sensitivity (64.3%) and specificity (100%) for areas of SD. CONCLUSION: GATA3 is a specific immunohistochemical marker for urothelial origin. CK14 is a highly specific and sensitive immunohistochemical marker of squamous cell carcinoma.

Prognostic utility of Ki-67 in gastric carcinoma

Introduction: Gastric carcinoma represents the third leading cause of cancer related mortality worldwide. Most of cases are diagnosed at the time of tumor metastasis. There is a discrepancy in the results regarding the role of Ki-67 proliferation index as a prognostic factor in gastric carcinoma. Objectives: To assess the prognostic value of Ki-67 proliferation index and correlate Ki-67 expression with the overall survival (OS) in patients with gastric carcinoma. Patients and Methods: This retrospective study included fifty cases collected randomly from archival paraffin blocks of gastric carcinoma. Cases were collected during the period from 2013 to 2018. Sections were prepared and the slides were immunohistochemically stained with Ki-67 antibody. Results: This study showed a statistically significant correlation between higher percentage of Ki-67 expression and cases with tumors located in the fundus and body of the stomach and with distant metastasis. Also a statistically significant correlation between higher mean of Ki-67 positive cell % and cases of adenocarcinoma with pT1 tumors and with loco-regional recurrence (P &lt; 0.001, P=0.02). A higher percentage of Ki-67 expression was found in cases of adenocarcinoma grade III and cases with positive perineural invasion compared to other cases, yet the correlation was statistically insignificant. According to survival analysis, OS rate was 34.0% and the median OS time was 14.6 months. There was no statistically significant association between Ki-67 proliferation index and disease free survival (DFS) and OS. Conclusion: Ki-67 proliferation index can be used as a predictor for distant metastasis and loco-regional recurrence of gastric cancer.

Abstract 835: T-cell abundance, clonality and disease specific survival in colorectal cancer

Abstract Immune responses to colorectal cancer (CRC) are important prognostic factors as measured by either tumor infiltrating lymphocytes per high powered field or the presence of Crohn's like lymphoid reaction. New technologies to quantify specific features of immune responses include immunoSeq (Adaptive Biotechnologies), which accurately quantifies T-cell receptor (TCR) abundance and T-cell receptor clonality. Here we measured the independent contributions of TCR abundance and TCR clonality to 5-year CRC-specific survival in the Molecular Epidemiology of Colorectal Cancer (MECC) study. The MECC study consented, interviewed, and followed 6,006 incident cases of CRC diagnosed between March 31, 1998 and July 1, 2017 (median follow-up time = 6.9 years). Archived paraffin blocks were retrieved, and 2,750 cases had sufficient tissue and adequate DNA extraction to perform the immunoSeq assay. Analyses were restricted to 1,625 samples with more than 100 T-cells to permit accurate quantification of TCR abundance and TCR clonality as measured by Simpson Clonality Index. TCR abundance was log2 transformed for analyses, and Simpson Clonality Index was log2-transformed and z-scale normalized separately by assay version. In unadjusted models, dichotomized log2 transformed TCR abundance (Hazard Ratio (HR) = 0.57, 95% confidence interval (CI) 0.47-0.71, p=9.77E-08) and dichotomized TCR clonality (HR = 0.77, 95% CI 0.63-0.94, p=0.01) were each statistically significantly associated with 5-year CRC-specific survival. In a model with adjustment for age, sex, microsatellite instability, stage and assay version, both dichotomized log2 transformed TCR abundance (HR = 0.61, 95% CI, 0.49-0.75, p=3.03E-06), and dichotomized TCR clonality (HR = 0.81, 95% CI, 0.66-0.99, p=0.048) were associated with improved 5-year disease-specific survival. Conclusion: TCR abundance and TCR clonality are independent prognostic indicators in CRC. Citation Format: Joseph Bonner, Sephanie L. Schmit, Sidney S. Lindsey, Ya-Yu Tsai, Rebeca Sanz-Pamplona, M. Henar Alonso, Marilena Melas, Hedy S. Rennert, Kevin J. McDonnell, Gregory Idos, Christopher P. Walker, W. Martin Kast, Diane Da Silva, Harlan S. Robins, Joel K. Greenson, Victor Moreno, Gad Rennert, Stephen B. Gruber. T-cell abundance, clonality and disease specific survival in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 835.

Methods for restoration of ki67 antigenicity in aged paraffin tissue blocks

Pathology archives are a treasure trove of paraffin embedded tissue spanning many years and covering a wide variety of tissues and diseases. The possibility of using old archival formalin fixed paraffin embedded (FFPE) tissues for diagnostic updates and research projects is a widespread need and it requires archives of stable, well-preserved samples. Immunohistochemistry performed on old archival paraffin blocks may give unreliable results, in particular for some antigens, such as Ki67. In consideration of this phenomenon, our aim is to comprehensively test and identify methods which may be used to obtain Ki67 immunohistochemical reactions of good quality from old archival FFPE blocks. Various methods were tested in order to evaluate their possible efficacy in increasing Ki67 immunointensity in a collection of 40-year-old, archival blocks including re-embedding, with deeper sectioning of tissue from the block and increasing heat-based pretreatment times (20 cases) and re-processing (20 cases). All reactions were performed using an automated immunostainer and Ki67 stained immunosections compared using a visual colour-based scale (the first immunostained section was considered as baseline). The combination of deep sectioning (1000 µM) and prolonged heat-based pretreatment (64 min) markedly increased immunoreactivity for Ki67. Re-embedding and reprocessing did not have a significant effect. Large tissue samples showed heterogeneity of Ki67 immunoexpression between the periphery of the sample and the central area. In conclusion, the study defines a useful protocol to increase antigen retrieval applicable to dated archival tissues.

Application of histochemistry AgNORs in diagnosing benign lesion, premalignant and malignant prostate tissue

Objective: Argyrophilic nucleolar organizer regions (AgNORs) count has been suggested as an objective method in differentiating of various non neoplastic lesions of the prostate. Material and Methods: This study was done on archival paraffin block consisting of 6 cases of each, benign, premalignant, and malignant lesions of the prostate. All specimens were studied and evaluated with Haematoxylyn and Eosin followed by AgNORs histochemistry staining and its expression was determined by counting AgNORs dot per nuclei in 100 nuclei using a 100x oil immersion lens. Results: The results showed a linear increasing trend of mean mAgNOR count according to prostatic lesion (benign to malignant). However, this is not statistically significance as the p-value > 0,05 (p- value=0,516). Conclusion: Based on this study, AgNOR cannot be use to determine prostatic lesion behavior. Futher study with larger samples should be done to get significant result.

Role of tesmin expression in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is the most commonly diagnosed cancer and the most frequent cause of cancer-associated mortality worldwide. Tesmin (MTL5) is a 60 kDa protein which has cysteine rich motifs, characteristic of metallothioneins. Tesmin expression was first observed in germ cells during spermatogenesis. Increased tesmin expression in NSCLC has been described previously. Minichromosome maintenance proteins (MCMs) serve a critical role in replication and cell cycle progression, i.e. in NSCLC. The aim of the present study was to evaluate the localization and intensity of tesmin, MCM5 and MCM7 protein expression in NSCLC and their association with the clinicopathological data of patients. Archival paraffin blocks of 243 cases of NSCLC and 104 non-cancerous tissue samples from the surgical margin (control) were obtained from patients treated at the Clinic of Thoracic Surgery of Wroclaw Medical University (Wroclaw, Poland) between 2010 and 2016, and were used for tissue microarrays and immunohistochemical (IHC) experiments. Laser capture microdissection was used for the isolation of cancer cells from 36 frozen samples of NSCLC and 8 control samples, and subsequently, MTL5, MCM5 and MCM7 mRNA expression was detected separately by reverse transcription-quantitative PCR. Positive cytoplasmic and nuclear tesmin, as well as nuclear MCM5 and MCM7 IHC expression were observed in 95.1, 83.67, 95.51 and 100% of the NSCLC cases, respectively. MTL5, MCM5 and MCM7 mRNA expression was observed in 91.66% of the cancer cases for all genes. The statistical analysis revealed increased tesmin IHC expression in cancer cells compared with the control. A positive correlation was observed between the IHC expression of nuclear tesmin and MCM5 proteins (r=0.33; P<0.0001) and nuclear tesmin and MCM7 proteins (r=0.315; P<0.0001). In addition, a positive correlation between the mRNA expression levels of MTL5 and MCM5 (r=0.421; P<0.05), MTL5 and MCM7 (r=0.557; P<0.01) was demonstrated. The survival analysis revealed that the presence of IHC cytoplasmic tesmin expression was a positive prognostic marker in NSCLC (P=0.0524). Furthermore, in vitro experiments performed on the NCI-H1703 cell line revealed that silencing of MTL5 mRNA and tesmin caused the downregulation of the expression levels of MCM5 and MCM7 and decreased the number of cells in the G2 phase. A positive association among tesmin, MCM5 and MCM7 could indicate a possible role of tesmin in the proliferation of NSCLC cancer cells.