Paraffin Blocks Of Tumors Research Articles

<i>CCND1</i> and <i>PTEN</i> genes as markers of progression in vulvar cancer

Purpose of the study. Studying the relative copy number of 12 genes: MYC, PTEN, CCND1, PIK3CA, TP53, CDKN2A, MDM2, MCL1, NFKBIA, MTAP, BIRC2, KMT2C, to search for potential prognostic cancer markers in vulvar cancer (VR).Patients and methods. The study included 50 patients aged 29–80 years with the diagnosis of squamous cell carcinoma of the vulva without metastases – group 1 (n = 25) and with metastases to the lymph nodes – group 2 (n = 25). Total DNA from paraffin blocks of tumor and conditionally healthy vulvar tissue served as the material for the study. The relative copy number of genes (RCQ) was assessed using the RT-qPCR method: MYC, PTEN, CCND1, PIK3CA, TP53, CDKN2A, MDM2, MCL1, NFKBIA, MTAP, BIRC2, KMT2C and referencegenes: GAPDH and B2M. Raw RT-qPCR data were analyzed using Bio-Rad CFX Manager software (ver. 2.1). RCQ of the genetic locus was calculated using the formula 2-ΔCt. Analysis of the significance of differences included determination of the OR (CI 95 %), Chi-square, Mann-Whitney U-test. Differences were considered statistically significant at p < 0.05.Results. Amplification was observed for 9 genes: MYC (48 % of cases), MCL1 (39 %), NFKBIA (46 %), CCND1 (52 %), PIK3CA (48 %), TP53 (62 %), MDM2 (40 %), BIRC2 (54 %), KMT2C (42 %); losses were recorded for 3 genes PTEN (44 % of cases), MTAP (32 %), CDKN2A (36 %). In the 2nd group of patients, a statistically significant 1.7-fold increase in RCQ of the CCND1 gene (p < 0.05) and a 1.9-fold decrease in RCQ of the PTEN gene (p < 0.05) were found.Conclusion. In the study, CCND1 amplification events and/or PTEN loss were associated with lymph node metastasis in RV patients. Thus, the copy numbers of these genes may serve as prognostic markers of the disease.

Comparison of 22C3-PD-L1 Expression Between Paired Tumor Paraffin Blocks of Surgical Resection Specimens of Nonsmall Cell Lung Cancer.

In this study, our aim was to evaluate the discordance of programmed cell death ligand 1 (PD-L1) expression between 2 paired paraffin blocks from surgical nonsmall cell lung cancer (NSCLC) specimens, thus providing recommendations for choosing paraffin blocks for PD-L1 immunohistochemistry detection. A total of 460 paired paraffin blocks of surgical NSCLC specimens were analyzed using the 22C3-PD-L1 assay. PD-L1 expression between 2 paired paraffin blocks was calculated using 3 classification schemes, tumor proportion score (TPS)-based 3-level classification and TPS-based binary classification using TPS ≥1% or TPS ≥50% as the cutoff. Clinicopathological characteristics were analyzed for cases with discordant PD-L1 expression. PD-L1 expression in 2 paired paraffin blocks of surgical NSCLC specimens was highly correlated from a single tumor ( R2 =0.89) in the overall trend. The overall discordance rates were 10.9%, 7.4%, and 3.5% respectively when using TPS-based 3-level classification or binary classification. No statistical differences were observed in PD-L1 expression discordance rates when patients were stratified by age, sex, smoking status, histologic types, TNM stage, or years of paraffin blocks ( P >0.05). The presence of a solid histologic pattern was associated with a higher PD-L1 expression discordance rate in adenocarcinomas ( P <0.05). Twenty-five cases with discordant PD-L1 expression were divided into 2 categories: spatial heterogeneity without recognizable morphology difference (76%) and spatial heterogeneity with significantly different morphology (24%). The discordance rate in TPS scores was much higher in cases with different morphology than those without different morphology. One representative paraffin block containing adequate neoplastic tissue may be adequate to determine PD-L1 expression in most of the surgical specimens of NSCLC. In cases that harbored different morphology in different paraffin blocks, the dual-block immunohistochemistry assessment method is recommended due to the intratumoral heterogeneity.

Expression of Cyclin D1 in Colorectal Cancer and Its Association With Clinicopathologic Parameters in Patients Underwent Colectomy

Colorectal cancer (CRC) is common cancer with a high mortality rate worldwide. Cyclin D1 is a gene that regulates cell cycle passage from stage G1 to S (G1/S checkpoint) and has recently been linked to the prognosis of a variety of cancers. Therefore, the aim of this study was to investigate the expression of cyclin D1 in colorectal cancers and its relationship with clinicopathologic factors. In this retrospective study, paraffin blocks of tumors of consecutive CRC patients registered in the histopathology laboratory of hospitals affiliated with Ahvaz Jundishapur University of Medical Sciences were used. Patients' clinicopathologic findings were extracted from their files, and using paraffin blocks, specific staining for cyclin D1 was performed using the immunohistochemistry method. Data were analyzed by SPSS software. In terms of staining, 11 samples (28.9%) scored 4, 11 samples (28.9%) scored 3, 8 samples (1/21%) scored 2, 3 samples (7.9%) scored 1, and 5 samples (2/13٪) scored zero. Staining intensity was severe in 10 cases (26.3%), moderate in 14 cases (36.8%), mild in 8 cases (21.1%), and negative in 6 cases (15.8%). The severity and extent of staining had no significant relationship with sex, age, tumor location, degree of differentiation (grade), depth of invasion, tumor size, lymph node involvement, and vascular and perineural invasion (P&gt;0.05). Despite the high expression of cyclin D1 in colorectal carcinoma, no significant relationship was observed between its expression and prognostic factors, which is probably due to the small sample size.

VEGF expression in cetuximab resistance development in patients with squamous cell carcinoma of the tongue and oral mucosa.

e18072 Background: Clinical course and aggressiveness of a tumor, as well as the subtle mechanisms of its pathogenesis are related to vascularization, which in turn is determined by the ability of transformed cells to synthesize a wide range of biologically active substances. Among modern approaches to cancer treatment, special attention is paid to anti-EGFR agents reducing tumor vascularization. The purpose of this study was to analyze VEGF expression in tumor vessels in patients with squamous cell carcinoma of the tongue and oral mucosa developing cetuximab resistance. Methods: The study included 45 patients with squamous cell carcinoma of the tongue and oral mucosa T3-4N0-1M0. The patients were divided into 3 groups: group 1 – all patients before treatment; group 2 – patients after standard chemotherapy with cisplatin and fluorouracil; group 3 – patients after chemotherapy in combination with targeted therapy with cetuximab. Immunohistochemical study was performed on sections from paraffin blocks of tumors using monoclonal murine anti-VEGF antibodies (clone VG1, Thermo Scientific) diluted 1:150. The Reveal Polyvalent HRP-DAB Detection System was used. The significance of differences was assessed using the Mann Whitney and Wilcoxon tests calculated using the STATISTICA 10.0 program (StatSoftInc., USA). Results: Changes in the density of vessels stained with the VEGF marker were studied in tumor cells of patients in all groups. Patients in group 1 had from 3 to 10 vessels in one field of view; in group 2 – from 2 to 6 vessels in 70% of cases (21) and up to 15 vessels in 30% of cases (9); in group 3 – only solitary vessels. The average numbers of microvessels stained with the VEGF marker per field of view were 6.5±1.52; 4.5±1.71; 1.0±0.28, respectively. Quantification of intratumoral microvascular density showed that the minimum number of microvasculature vessels in the field of view was observed in patients who received chemotherapy and targeted therapy with cetuximab. The value was 5 times lower compared to initial values, and 4 times lower than in patients who received only standard chemotherapy (the Mann Whitney test, p &lt; 0.05). The statistical analysis according to the Wilcoxon test on the effect of targeted therapy (comparison between groups 2 and 3) revealed a statistically significant effect of cetuximab for this marker (p = 0.028). Conclusions: Immunohistochemical analysis of the VEGF neoangiogenesis factor in squamous cell carcinoma of the tongue and oral mucosa confirmed the effectiveness of targeted therapy with cetuximab.

Characteristics of expression of cancer stem cell markers in various approaches to cervical cancer treatment.

e17516 Background: Cancer stem cells (CSCs) are being actively studied as a possible origin of malignant neoplasms. Research of the properties of CSCs and mechanisms of their regulation holds the promise of the development of new approaches to antitumor therapy. The most frequently detected CSC markers in different tumors are CD44 and CD133. In this regard, the aim of our study was to identify characteristics of expression of CSC markers in cervical cancer treatment and their possible predictive value. Methods: The study included 237 patients with locally advanced cervical cancer T2a-2bNx-1M0. Patients were divided depending on the treatment: neoadjuvant chemotherapy with cisplatin and bleomycin (NACT, n = 84); plasmapheresis (PA) in combination with NACT (PA+NACT, n = 60); PA+NACT with nonspecific immunotherapy with an interferon inducer (PA+NACT+IT, n = 93). Immunohistochemical study was performed on sections from paraffin blocks of tumors using anti-CD44 monoclonal mouse antibodies (156-3C11, Thermo Scientific) at a dilution of 1:2500 and anti-CD133 polyclonal rabbit antibodies (AF5120, Affinity Biosciences) at a dilution of 1:400 (Thermo autostainer Scientific 480S). Membrane staining and staining intensity were evaluated: 0, 1+ weak, 2+ moderate, 3+ strong staining. Protein expression was defined as positive when staining was detected in ≥10% of all tumor cells with a staining intensity ≥2. Statistical analysis of the results was performed with the STATISTICA 13.0 program (StatSoftInc., USA) using the Mann-Whitney U-test. Results: Tumors with positive CD133 and CD44 expression were observed in all groups of patients. However, only PA+NACT+IT patients had CD133- (57%) and CD44- (43%) tumors. Median expression of CD133: in NACT - 70 [60;80]; PA+NACT - 60 [37;70], PA+NACT+IT - 25 [5;60]. Median expression of CD44: in NACT - 30 [10;40]; PA+NACT - 30 [13;65], PA+NACT+IT - 20 [2; 40]. CD133 expression in PA+NACT+IT patients was significantly lower than in PA+NACT and NACT patients by 2.4 times (*p = 0.009) and 2.8 times (*p = 0.002), respectively. CD44 expression in tumors of patients with PA+NACT+IT was 1.5 times lower (statistically non-significant). Differences in tumors of NACT and PA+NACT patients were not found. Conclusions: The immunohistochemical study revealed a decline in the expression of CSC markers (CD133 and CD44) in tumors of patients who received PA+NAPCT+IT. The results obtained for the CD133 marker show its possible value as a predictive factor in the treatment of cervical cancer.

Desmocollin 3 (DSC 3) expression in different subtypes of non-small cell lung cancer.

e20513 Background: Currently, an active search is underway for additional markers that predict the risk of tumor progression based on the epithelial-mesenchymal transition (EMT). Desmocollin 3 (DSC 3), a member of the Cadherin superfamily and a cell adhesion marker, is one of the markers for this transition. The aim of our study was to evaluate the expression of DSC 3 as a prognostic factor in different subtypes of non-small cell lung cancer. Methods: The study included 12 patients with squamous cell lung cancer and 8 patients with lung adenocarcinoma, aged 49 to 76 years. Immunohistochemical study was performed on sections from paraffin blocks of tumors using polyclonal rabbit antibodies to Desmocollin 3 (Invitrogen) at a dilution of 1:300 and the UltraVision Quanto Detection System HRP DAB. The expression of DSC 3 was semiquantitatively analyzed according to the percentage of cells and the intensity of staining: 0; 1+ (weak); 2+ (moderate); 3+ (high). Expression was considered positive if the score was ≥2. The Mann-Whitney U-test, Pearson's χ² distribution, and Spearman's correlation coefficient were used for statistical analysis of the results. Results: The predominance of patients with DSC3+ tumors was noted in the group with squamous cell carcinoma (64%), while the majority of patients with adenocarcinoma had DSC3- tumors (71%). However, Pearson's χ² was not statistically significant. Differences in DSC 3 expression values by 1.3 times were insignificant (p = 0.082): Me = 40 [0-80] in squamous cell carcinoma, Me = 30 [0-65] in adenocarcinoma. The correlation analysis between the expression of this marker and the survival rate of patients also had no statistical significance (rs = 0.061 for squamous cell carcinoma and rs = 0.196 for adenocarcinoma, p &gt; 0.05). Conclusions: The immunohistochemical study revealed some specific features in the expression of DSC 3 in different subtypes of non-small cell lung cancer.

Expression of markers of epithelial-mesenchymal transition in various forms of vulvar cancer.

e17532 Background: Vulvar cancer is a rare gynecologic tumor accounting for 2–5% of all gynecologic cancers. The epithelial-mesenchymal transition (EMT) is considered one of the factors contributing to the aggressive behavior of vulvar squamous cell carcinoma. E- and N-cadherins are key regulators of this transition. The purpose of our study was to identify the features of the expression of EMT markers in vulvar cancer as prognostic factors. Methods: The study included 30 patients aged 39-70 years, mean age 52 years, with vulvar cancer: control group (n = 15) with vulvar cancer T1-2N0-M0 (G2); main group (n = 15) with vulvar cancer T1-2N1-M0 (G2) with metastases to inguinal lymph nodes. Immunohistochemical study was performed on sections from paraffin blocks of tumors using monoclonal mouse antibodies to E-cadherin (12#, Cloud-Clone Corp.) and N-cadherin (389, Invitrogen) and the UltraVision Quanto Detection System HRP DAB. Expression of E- and N-cadherin was semiquantitatively analyzed according to the cell percentage and stain intensity: 0 (0-10%); 1+ (11-30%); 2+ (31-70%); 3+ (&gt; 70%). Expression was considered positive if the score was ≥2. Pearson's χ² test and Spearman's correlation coefficient were used for statistical analysis. Results: The maximum staining for E-cadherin was observed in the control group (in 73%), for N-cadherin - in the main group (in 59%). Median expressions of E- and N-cadherin differed: in the control group, 70 [40-78] and 26 [0-40]; in the main group, 47 [45-60] and 59 [27-65]. Pearson's chi-squared test showed that patients with E-cadherin+ tumors (p = 0.006) statistically significantly prevailed in the control group, and patients with N-cadherin+ tumors (p = 0.027) prevailed in the main group. However, when considering the correlation between E-cadherin and N-cadherin in the groups, no statistically significant differences were found (rs = -0.042 in the control group and rs = 0.250 in the main group at p &gt; 0.05), apparently due to the limited sample. Conclusions: The immunohistochemical study revealed a tendency towards loss of E-cadherin and increased expression of N-cadherin, which is often associated with poor prognosis.

Relationship of E-cadherin, Beta-catenin, N-cadherin, ZEB1 and αSMA as Epithelial Mesenchymal Transition markers with prognostic factors in early and advanced stage laryngeal squamous cell carcinomas.

To investigate the relationship between E-cadherin, beta-catenin, N-cadherin, ZEB1, and αSMA as epithelial-mesenchymal transformation markers with tumor stage, lymph node metastasis (LNM), and overall survival (OS) in laryngeal squamous cell carcinomas (LSCC). A total of 100 cases diagnosed with LSCC were included in the study. Data about the lymphovascular invasion (LVI), perineural invasion (PNI), necrosis, and LNM were recorded by evaluating hematoxylin-eosin-stained slides. Markers of E-cadherin, beta-catenin, N-cadherin, ZEB1, and αSMA were applied to the sections prepared from paraffin blocks of tumor samples. Ninety-five male and five female patients were included in the study, and 38 of them exited. A significant relationship was observed between OS with advanced tumor stage, presence of LNM and PNI. A significant relationship was found between increased tumor Zeb1 expression and advanced tumor stage. In univariate and multivariate analyses, a significant negative relationship with OS, and increased Zeb1 expression in tumor and tumor stroma was seen. Any relationship was not observed between E-cadherin, beta-catenin, N-cadherin, and αSMA and OS. Among the EMT markers, we evaluated in our study, it was seen that Zeb1, which is an EMT transcription factor, is associated with tumor stage, LNM, and OS. Remarkably, Zeb1 expression observed in tumor stroma was also significant for OS. Any similar data reported for LSCCs have not been encountered in the literature, and it was thought that it would be appropriate to support our findings with further studies to be performed on this subject.

Prognostic value of desmoplastic stroma in intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinomas (iCCs) are primary tumors of the liver characterized by the presence of a desmoplastic stroma. While tumor stroma may have a protective or a pejorative value depending on the type of malignant disease, the precise role of the desmoplastic stroma in iCC remains poorly understood. The aim of the present study was to evaluate the prognostic value of stromal compartment in iCC through a multiparametric morphological analysis. Forty-nine surgically resected iCCs were included. For all cases, tumor paraffin blocks of iCCs were selected for stromal morphological characterization through quantitative and qualitative approaches using immunohistochemistry and second-harmonic generation imaging. Intratumor heterogeneity was also evaluated in regards with the different stromal features. High proportionated stromal area (PSA) (defined by stromal to tumor area ratio) was inversely correlated with vascular invasion (62.5% vs 95.7%, p = 0.006) and positively correlated with well-differentiated grade (60% vs 12.5%, p = 0.001). Patients with high PSA had a better disease-free survival (DFS) than patients with low stromal area (60% vs 10%, p = 0.077). Low activated stroma index (defined by cancer-associated fibroblasts number to stromal area ratio) was associated with a better DFS (60% vs 10%, p = 0.05). High collagen reticulation index (CRI), defined as the number of collagen fiber branches within the entire length of the collagen network, was associated with a poorer overall survival (42% vs NR, p = 0.026). Furthermore, we showed that CRI was also an homogeneous marker throughout the tumor. Based on morphological features, desmoplastic stroma seems to exert a protective effect in patients with iCC. Stromal collagen reticulation may provide additional clinically relevant information. In addition, these data support the potential value to evaluate CRI in biopsy specimen.

Prevalence of Tissue BRCA Gene Mutation in Ovarian, Fallopian Tube, and Primary Peritoneal Cancers: A Multi-Institutional Study.

Background and objective:Ovarian, fallopian tube, or primary peritoneal cancer patients with BRCA gene mutation have enhanced sensitivity to platinum-based regimens and PARP inhibitors. However, the knowledge regarding BRCA mutation in Thai patients is limited. This study aimed at identifying the prevalence and characteristics of somatic and germline BRCA 1 and 2 mutations in Thai patients with these cancers. Materials and Methods:The paraffin blocks of tumors with histology of high grade serous, high grade endometrioid, or clear cell carcinoma obtained between June 2016 and December 2017 were analyzedto evaluate BRCA mutation using next-generation sequencing system. Blood or normal tissue paraffin blocks of positive patients were further tested for germline BRCA mutation. Results:Tissue paraffin blocks of 178 patients were collected but only 139 were analyzed. Positive BRCA mutation was identified in 24 patients (17.3%): BRCA1 in 13 cases, BRCA2 in 10 cases, and BRCA1 and 2 in the rest one. Germline mutation study in blood or normal tissue in 23 positive patients revealed BRCA mutation in 14 cases, BRCA1 in 8 cases and BRCA 2 in 6 cases. Overall, the prevalence of somatic and germline mutation was 6.5% (9 out of 138 patients) and 8.7% (14 out of 138 patients), respectively. The most common histology associated with BRCA mutation was high grade serous cancer (27.3%). No significant difference was found between patients with or without BRCA mutation in terms of stage, outcome, platinum status, and survival outcome. Conclusion: BRCA mutation was demonstrated in less than 10% of Thai ovarian cancer patients. Higher rate of mutation was found in high grade serous cancer.

Expression of CD44 and CD133 markers in regional metastases from breast cancer.

e13087 Background: High incidence and mortality of breast cancer (BC) require a constant search for the most informative and effective methods of diagnosis and treatment evaluation. The purpose of the study was to analyze phenotypic characteristics of cancer stem cells in tumor tissues and regional metastases of breast cancer (BC). Methods: The study included 20 BC patients with regional metastases (lymph nodes) aged 32-74 years, mean age 56.1±3.3 years. IHC analysis was performed on sections from paraffin blocks of tumors using monoclonal mouse antibodies to CD44 (156-3C11 Thermo Scientific) at a dilution of 1:2500 and polyclonal rabbit antibodies to CD133 (Cloud-Clone Corp.) at a dilution of 1:700 using the Thermo Scientific automated stainer. Membrane staining and staining intensity were evaluated: 0, 1+ weak, 2+ moderate, 3+ strong staining. CD44 expression was considered positive when staining was detected in more than 10% (cut-off) tumor cells. CD133 expression was considered positive when staining was detected in more than 5% (cut-off) of the tumor. Results: CD44 + expression was detected in 80% of breast tumor tissues (16 samples), while in 20% this marker was not determined. The average CD44 expression was 27.2±13.9%. The expression of CD44 in regional metastases (RM) from BC was 35% (from 0 to 30%), on average 17.0±6.8%. When distributed according to the χ2 criterion in the tumor and RM, an association between factorial and resultant signs was statistically significant (8.286, at p &lt; 0.004). CD133+ expression in tumor tissues was detected in 80%, with an average expression level of 56.0±14.6%, with a predominance of positive cytoplasmic staining in 60%. Determination of this marker in BC RM gave a positive result in 15% (3 samples), with expression fluctuations from 0 to 60%, the average level was 33.0±15.4%. When distributed according to the χ2 criterion in the tumor and RM, an association between factorial and resultant signs was statistically significant (16.942, at p &lt; 0.001). Conclusions: Determination of tumor cells with the stem phenotype directly in the tissue of primary BC tumors and in tissues of regional metastases demonstrated that the percentage of CD44+ and CD133+ cells in the primary BC tumor was higher.

Importance of MACC1 expression in breast cancer and its relationship with pathological prognostic markers.

Metastasis associated colon cancer gene 1 (MACC1) is a gene that was first described as a c-Met transcription regulator causing the progression of colon cancer. In this study, protein and messenger RNA (mRNA) expression of MACC1 in breast cancer and its relationship with clinicopathological prognostic parameters were investigated. Sixty-six cases with tumors underwent radical mastectomy for invasive ductal carcinoma and 25 control cases operated for mammoplasty were included in the study. In paraffin blocks of tumor and control tissues, MACC1 expression was investigated by the immunohistochemical method and Real-time polymerase chain reaction (Real-Time PCR). In addition, vascular endothelial growth factor (VEGF) expression was examined immunohistochemically in tumor tissues. The relationship between MACC1 expression in tumor tissues, clinicopathological prognostic parameters, and VEGF was investigated. In this study, protein and mRNA expressions of MACC1 were found to be higher in tumor tissues compared with normal breast tissues. MACC1 protein expression was also associated with significant poor prognostic markers, such as high histologic grade, ER negativity, and HER2 positivity. However, there was no correlation between MACC1 expression and VEGF. According to these results, MACC1 expression may be a marker of breast carcinoma as well as an independent predictor of poor prognosis. In addition, MACC1 may not affect angiogenesis in breast cancer or even if it has an effect, it may not be associated with VEGF. However, it would be appropriate to support these results in a larger series by investigating in vivo and in vitro studies.

КЛИНИЧЕСКИЕ ОСОБЕННОСТИ ТЕЧЕНИЯ КОЛОТЕРАЛЬНОГО РАКА У ПАЦИЕНТОВ С МУТАЦИЯМИ EGFR-СИГНАЛЬНОГО ПУТИ

KRAS, NRAS, BRAF mutations are associated with an unfavorable prognosis for colorectal cancer (CRC). At the same time, there is no single point of view on disease development during adjuvant chemotherapy (ACT). Objective. The authors aimed at studying KRAS, NRAS and BRAF mutations in the tumor and their influence on the clinical characteristics of colorectal cancer development. Materials and Methods. Paraffin blocks of the primary CRC tumor (n=37) were used as the material for the study. Using genomic DNA, isolated from the primary tumor, real-time PCR was used to determine the most common mutations in CRC: KRAS gene (exon 2, codon region 12–13), NRAS gene (exon 3, codon region 61), B6F V600E gene. Results. The results of genotyping of DNA samples isolated from the primary CRC tumor paraffin blocks showed that BRAF gene mutations were detected in 8.2 % of cases, NRAS gene mutations were detected in 5.4 % of cases, and KRAS gene mutations were detected in 37.8 % of cases. The authors didn’t reveal any dependencies of the mutation distribution on patients’ gender and age. The examined mutations were more common in adenocarcinomas of high and moderate degrees of differentiation. The relapse-free period after ACT in patients with identified KRAS, NRAS, BRAF gene mutations is significantly less than in those without mutations. Conclusion. The findings suggest that EGFR signaling pathway mutations (KRAS, NRAS and BRAF) increase the risk of disease recurrence and are an unfavorable prognostic factor. Keywords: colorectal cancer, NRAS, KRAS, BRAF mutations, adjuvant chemotherapy.

КЛІНІЧНІ ОСОБЛИВОСТІ ІМУНОГІСТОХІМІЧНОЇ ДІАГНОСТИКИ САРКОМ ТА ФІЛОЇДНИХ ПУХЛИН ГРУДНОЇ ЗАЛОЗИ

Мета дослідження – вивчення імуногістохімічних особливостей сарком та філоїдних пухлин грудної залози.Матеріали та методи. Для ІГХ-дослідження було відібрано 60 парафінових блоків пухлин, у яких гістологічно було верифіковано діагноз саркоми (42) та філоїдної пухлини (18). ІГХ-дослідження проводили з використанням спектра відповідних антитіл, який включав маркери панцитокератинів (клон AE1/AE3); ERG (клон EP111); SOX-10 (клон EP268); TLE1 (клон 1F5); кальдесмону (клон h-CD); міогеніну (Myf-4) (клон F5D); MyoD1 (клон EP212); десміну (клон D33); MDM2 (клон 1B10); CDK4 (клон EP180); CD68 (клон PG-M1); CD34 (клон QBEnd 10); CD31 (клон JC70A); бета-катеніну (клон betaCatenin-1); гладеньком’язового актину альфа (клон 1A4); актину (клон HHF35); епітеліального мембранного антигену (EMA, MUC1) (клон E29). Також визначали рівень експресії маркера проліферації Ki-67 (клон MIB-1). Дослідження проводили в лабораторії CSD Health Care, м. Київ.Результати дослідження та їх обговорення. Після проведених імуногістохімічних досліджень виявилось, що із 12 сарком без уточненого гістотипу в 9 випадках були плеоморфні саркоми, у 2-х – міофібробластні саркоми, та одна остеосаркома. Із 27 блоків ангіосарком імуногістохімічно було підтверджено 17 (63,0 %), інші 10 (37,0 %) виявились карциномами. Фібросаркоми (три гістологічні блоки) були повністю підтверджені імуногістохімічно. Серед 18 гістологічних блоків філоїдних пухлин проміжного типу у двох випадках (11,1 %) було встановлено злоякісний варіант ФП.Висновки. Імуногістохімічні дослідження відіграють важливу роль у диференційній діагностиці і визначенні гістотипу СГЗ та встановленні точного діагнозу при ФП. Запропоновано діагностичний алгоритм, що включає необхідні ІГХ-маркери в якості базовової панелі для ІГХ-дослідження, як у випадку СГЗ, так і при злоякісних ФП.

HER2 mRNA expression in breast cancers with equivocal immunohistochemical results using in situ mRNA hybridization

To investigate in situ mRNA expression of HER2 oncogene in breast cancers with equivocal immunohistochemical results, and to explore the potential feasibility of RNAscope technique in evaluating HER2 status in breast cancers. Sixty-nine FFPE samples of invasive ductal breast cancer with equivocal HER2 immunohistochemistry results (IHC 2+) were collected from surgical excisions from Peking Union Medical College Hospital between June 2010 and June 2013. HER2 status and in situ mRNA expression were tested by fluorescence in situ hybridization (FISH) and RNAscope respectively using tissue microarray constructed from tumor paraffin blocks. The results of HER2 mRNA expression were scored 0 to 4 (from low to high levels) according to mRNA expression in 100 cancer cells. HER2 mRNA expression was evaluated in two groups of patients, with positive and negative FISH results. Twenty-three of the 69 samples were FISH positive, including 16 samples that were scored 4 by RNAscope (70%, 16/23), 6 samples were scored 3 (26%, 6/23) and one sample was scored 2 (4%, 1/23). High in situ mRNA expression (score 4 or 3) were observed in 96% of HER2 FISH positive samples. All of samples that were scored 4 by RNAscope were FISH positive. Forty-six samples were FISH negative, including 17 samples that were scored 3 by RNAscope (37%, 17/46), 25 samples were scored 2 (54%, 25/46), and 4 samples were scored 1 (9%, 4/46). Breast cancer with HER2 IHC 2+ could be further classified according to in situ mRNA expression status. Among them, RNAscope score of 4 could be one of the interpretation criteria for re-testing IHC 2+ samples. In situ detection of HER2 mRNA may be an additional candidate method of confirmation for HER2 gene amplification or protein overexpression, and has potential clinical utility.

The Relationship Between IMP3 Expression in Colorectal Adenocarcinoma and Clinicopathologic Findings

Background: The IMP3 is an oncofetal protein, which has been recently proposed as a diagnostic and prognostic marker in many cancers, including colorectal adenocarcinoma. The overexpression of IMP3 seems to have a correlation with patient’s prognosis. Objectives: In this study, the relationship between IMP3 expression in colorectal adenocarcinoma and clinicopathologic findings was assessed. Patients and Methods: In this study 112 colorectal tumor paraffin blocks of Rasoul-e-Akram hospital were stained for IMP3 and slides were assessed for intensity and extent of positivity. The statistical relationships between marker expression and clinicopathologic findings (degree of differentiation, tumor size, depth of invasion and lymph nodes metastasis) were assessed. Data were analyzed by the SPSS 21 software and logistic regression and chi-square test, with p-values of less than 0.05 indicating statistical significance. Results: Immunoreactivity pattern of IMP3 was cytoplasmic in different clinicopathologic findings. Among different clinicopathologic findings, we found a statistical relationship between tumor differentiation and IMP3 (P = 0.047); so that, the poorly differentiated tumors were positive for this marker. No relationship was found between tumor size, depth of invasion or lymph node involvement and IMP3. Conclusions: IMP3 immunoreactivity was associated with poor differentiation of tumor yet not associated with tumor size depth of invasion or lymph node involvement

Prognostic values of ETS-1, MMP-2 and MMP-9 expression and co-expression in breast cancer patients

The aim of this study was to analyse expression of ETS-1 protein and two gelatinases (MMP-2 and MMP-9) and their possible prognostic value in breast carcinoma patients, as well as correlation of their expression with other known prognostic factors such as tumor size, grade, vascular invasion, steroid receptor values, HER2 values and proliferative index. The expression of MMP-2, MMP-9 and ETS-1 was immunohistochemicaly analysed in 121 consecutive primary breast carcinoma patients who underwent surgery at the Clinical Hospital Centre Zagreb during 2002. Three representative areas from each tumor paraffin blocks were taken and arranged on a recipient paraffin block with predefined coordinates for simultaneous analyses of multiple tissue samples (TMA). ETS-1, MMP-2 and MMP-9 expression and co-expression were correlated with other clinico-pathological parameters and based on the available clinical follow up data survival analysis was performed. The ETS-1 protein is found to be expressed in tumor cell nuclei and cytoplasm as well as in stromal lymphocytes, fibroblasts and endothelial cells. MMP-2 and MMP-9 were found to be expressed in cytoplasm of both, tumor and stromal cells. For our analysis only tumor cell expression was used for statistical analysis. We found 56,2% ETS-1 positive tumors, 77,7% were MMP-2 positive, and MMP-9 was expressed in 90% of primary breast carcinomas. There were no significant correlations between MMP-s expression and other patohistological prognostic factors, but expression of ETS-1 was significantly correlated with higher tumor size and grade, as well as with negative steroid receptors. Co-expression of MMP-2, MMP-9 and ETS-1 was found in 40,5 % of tumors, and more commonly was found in tumors larger than 2 cm, high grade tumors, and steroid receptor negative tumors. In univariate analysis, statistically significant negative impact on overall survival (OS) had tumor size, nuclear and tumor grade, ETS-1 expression in tumor cells, co-expression of ETS-1 either with MMP-2 or MMP-9, as well as co-expression of ETS-1, MMP-2 and MMP-3. Disease free survival (DFS) was significantly shorter in patients with tumors greater than 2 cm, ETS-1 positive tumors, ETS-1 and MMP-2 or MMP-9 co-expressed tumors, and additionally in tumors with ETS-1, MMP-2 and MMP-9 co-expression. These results suggest that expression of ETS-1 as well as MMP-2, MMP-9 and ETS-1 co-expression might be used as a poor prognostic factor in breast cancer patients.

Association of Epstein-Barr virus infection and breast carcinoma.

IntroductionA controversy regarding the association of Epstein-Barr virus (EBV) with breast carcinomas has recently been reported in the literature. The present study was carried out in an attempt to determine whether there is a relationship between latent infection with EBV and breast carcinomas in Jordanian females.Material and methodsExtraction of DNA from the archive samples of breast carcinoma cases embedded in paraffin wax was performed and the extracted DNA was subjected to polymerase chain reaction amplification to detect the EBV genome using four sets of primers for EBER 2, BNLF-1, EBNA 2, and Gp220. Immunohistochemistry study was performed on sections of 4 µm which were cut from paraffin blocks of tumor and control groups. Monoclonal antibody against EBNA-1 was applied to all slides to identify the EBV-infected tumor cells. Detection was performed using the Dako envision dual link system.ResultsDNA was successfully extracted from 92 paraffin embedded samples of breast carcinoma patients, and from 49 normal samples. The extracted DNA was confirmed by using glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) primers. Twenty-four out of 92 breast carcinoma specimens was found to be infected with EBV as compared to 3 out of 49 control group specimens, which represented a statistically significant difference (p-value using χ2 = 0.008). Immunohistochemically, 24 (26%) of the 92 studied samples were found to be positive, showing EBNA-1 granular nuclear staining in tumor epithelial cells.ConclusionsThese findings suggest an association between EBV infection and breast carcinoma development.

Abstract 867: Somatic BRAF and NRAS mutations in familial melanoma: a GenoMEL study

Abstract Up to 10% of all melanoma patients show a family history of disease. One main predisposing gene for familial melanoma development is CDKN2A, a tumor suppressor gene that encodes two distinct proteins, p16INK4A and p14ARF. CDKN2A germline mutations have been found in 20-40% of melanoma-prone families. So far, BRAF and NRAS are the most frequently altered oncogenes described in cutaneous melanoma. To date, little is known about the somatic changes that occur in familial melanoma. The aim of this study was to determine the prevalence of BRAF and NRAS somatic mutations in familial melanomas and to evaluate the associations between these mutations and clinicopathological factors. The study was performed as a collaboration within GenoMEL (The Melanoma Genetics Consortium). A total of 135 primary familial cutaneous melanomas (88 tumors from CDKN2A mutation-carriers and 47 from CDKN2A non-carriers) were investigated. Paraffin-blocks of tumors were collected from 8 GenoMEL centers in Australia, Italy, Latvia, the Netherlands, Spain, Sweden, and UK. Tumor cells were isolated by laser capture microdissection and mutation screening (BRAF exon 15 and NRAS exon 3) was performed by direct sequencing. Mutation analysis revealed BRAF and NRAS mutations in 41.6% and 11.4% of tumors, respectively. In no case were BRAF and NRAS mutations found to coexist in the same tumor. There was no difference in BRAF mutation frequency between tumors from patients with CDKN2A alterations and tumors from patients wild-type for CDKN2A (41.0% vs. 42.9%). Similarly, the frequency of NRAS mutations did not differ markedly when tumors from CDKN2A mutation-carriers and non-carriers were compared (9.0% vs. 15.6%). There was also no difference in the frequency of BRAF mutations between tumors with CDKN2A mutations disrupting both p16INK4A and p14ARF and tumors with CDKN2A mutations affecting p16INK4A only (40.6% vs. 36.4%). When tumors with different CDKN2A founder mutations were compared, BRAF mutations were seen in 50.0%, 45.8%, 37.5% and 35.0% of p.M53I, p.R112_L113insR, p.G101W and c.225_243del19 altered tumors, respectively. NRAS mutations were observed in 14.3% 12.5% and 10.5% of p.R112_L113insR, p.M53I and c.225_243del19 altered tumors, respectively, whereas no NRAS mutations were seen in tumors with the p.G101W founder mutation. Patients with BRAF-mutated tumors had a significantly lower median age at diagnosis compared to patients with NRAS-mutated or BRAF/NRAS wild-type tumors (43.5, 54 and 51.5 years, respectively). Moreover, melanomas with mutated BRAF or NRAS were thicker than BRAF/NRAS wild-type cases (median thickness 1.0, 1.4 and 0.7mm, respectively). Our results suggest a role for the BRAF and NRAS oncogenes in familial melanoma. Somatic changes of these genes occur in tumors from both CDKN2A mutation carriers and non-carriers. Presence of BRAF or NRAS mutations associated with patient and tumor characteristics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 867. doi:10.1158/1538-7445.AM2011-867

PROTEOMIC PROFILES OF DESMOID TUMORS AND OSTEOSARCOMA OF BONE AND SOMATIC SOFT TISSUE

DESMOID TUMORS AND HIGH GRADE OSTEOSARCOMAS OF BONE AND SOMATIC SOFT TISSUE HAVE DISTINCT PROTEOMIC PROFILESProteomic analysis of recurrent but non metastasizing desmoid tumors, and osteosarcoma (OS) with known metastatic potential occurring in older individuals was performed to identify potential predictive marker proteins for drug resistance and prognosis. Archived samples from nineteen desmoid tumors and fourteen primary osteosarcoma occurring in soft tissue, bone were collected following IRB approval. Five micron thick sections were obtained from paraffin blocks of tumor and scraped, digested and analyzed by mass spectrometry. Protein abundances determined by spectral counting and used for hierarchical cluster analysis. Increase in HSP70, clusterin, emelin, creatine kinase B and carbonic anhydrase noted predominantly in OS. Results were validated for myoglobin and clusterin by immunohistochemical stains.. Proteins at higher abundance in OS relate to oxidative stress and hypoxia. HSP90 and clusterin may be markers of aggressive extraosseous osteosarcoma and may be indicative of drug resistance. The absence of these proteins in desmoids support their being considered benign locally recurrent tumors.