The Importance of Immunohistochemical Heterogeneous Expression of MMR Protein in Patients with Colorectal Cancer in Stage II and III of the Disease.

Background and objectives: In patients with colorectal cancer (CRC), heterogeneous expression of Mismatch repair (MMR) proteins can manifest itself in several different forms and is not such a rare phenomenon. Therefore, it is very important to recognize the nuclear expression of MMR proteins of different MMR status in order to avoid false positive or false negative results. The aim of this study was to determine the frequency and distribution of heterogeneous expression of MMR proteins in patients with stages II and III of the disease as well as its association with clinical, demographic and pathological characteristics of CRC in relation to proficient and deficient expression of MMR proteins. Material and Methods: The study included 104 cases of colorectal cancer obtained from surgical colectomy material in stages II and III of the disease. Results: From a total of 104 patients with colorectal cancer, immunohistochemical analysis of the expression of all four MMR proteins showed that heterogeneous expression of MMR proteins (as well as deficient immunoreactivity of tumor cells) was present in 12 cases, while proficient expression of MMR proteins was detected in 80 tumors. Conclusions: Our study showed that the only independent predictors of the loss of MMR protein expression were younger patient age and right-sided anatomical location of the tumor. The study also established the existence of heterogeneous expression of MMR proteins in a non-negligible percentage of CRCs (11.5%), where heterogeneous nuclear expression of MMR proteins was described in several different forms.

Prognostic Value and Immunohistochemical Analysis of Mismatch Repair Deficiency in Patients with Stage II and III Colorectal Carcinoma-A Single-Center Study.

Background and objectives: Deficient mismatch repair (MMR) status is associated with good prognosis but poor therapeutic response to adjuvant chemotherapy in patients with colorectal cancer. However, there are some opposed arguments considering therapeutic outcomes in patients with evidenced MMR deficiency in colorectal cancer. The aim of the study was the investigation of prognostic value and immunohistochemical analysis of the MMR-deficiency tumors. Materials and Methods: The study enrolled 104 patients with resected stage II and III colorectal cancer samples from the period 2018-2019. Results: The tumors with deficient MMR status were significantly associated with age up to 50 years and right-sided localization (p < 0.001). During the follow-up period of 22.43 ± 6.66 months, 21 patients (20.2%) died, whereas 14 patients (13.5%) had relapses. The loss of mutL homologue 1/postmeiotic segregation increased 2 (MLH1/PMS2) expression, compared to proficient MMR tumors, was associated with shorter disease-free survival in patients with lymphovascular invasion (p < 0.05), perineural invasion (p < 0.01), stage III (p < 0.05) and high-grade tumor (p < 0.05). Conclusions: This retrospective pilot study of a single-center cohort of patients with stage II and III colorectal cancer highlights the clinical importance of using immunohistochemistry (IHC) analysis as a guide for diagnostic algorithm in a country with limited resources, but with a high prevalence of colorectal carcinoma in the young patients. MMR-deficiency tumors compared with proficient MMR colorectal cancer was not shown to be a significant predictor of disease-free and overall survival.

Association of axillary node status with clinicopathological characteristics and expression of EZH2 and CD44 in primary breast ductal carcinoma.

OBJECTIVE: In order to enhance the prognostic benefit of new molecular markers, the aim of this study was to identify possible association of axillary lymph node (ALN) status and pN with clinicopathological characteristics and expression of EZH2 and CD44 in invasive ductal carcinoma (IDC) of the breast. METHODS: The investigation included 106 patients with IDC who had undergone radical mastectomy at the Clinic of Endocrine Surgery in Nis. Clinicopathologic parameters and immunohistochemical expression of EZH2 and CD44 in primary IDC were investigated in relation to ALN status and pN. RESULTS: Our univariate analysis established that T3-T4 stage, high EZH2, and high EZH2 with ER- were associated with ALN metastasis (p=0.014; 0.003; 0.013). Decreased probability for ALN involvement was found with T1 stage, and low EZH2 with ER+ (p=0.032; 0.022). Multivariant analysis established that high EZH2 in cancer cells was associated with high risk for ALN metastases (p=0.004); T1 tumors were associated with low risk (p=0.037). Higher pN was associated with high EZH2, high EZH2 with ER-, as well as an advanced clinical and disease stage (p=0.006; 0.001; p=0.002, 0.001). Lower pN was associated with ER+, and ER+ with low EZH2 (p= 0.004; 0.012). CD44 was not associated with ALN involvement, nor with pN. CONCLUSIONS: This study revealed association of EZH2 with ALN metastases, where disease stage and expression profiles of EZH2 and ER may have affected regional pN.

Interlaboratory concordance in HER2 testing: Results of a Serbian ring-study.

PURPOSE: The purpose of this study was to assess the immunohistochemistry and chromogenic in situ hybridization (CISH) inter-laboratory consensus between national pathology laboratories in Serbia. METHODS: This study was conducted between 2013 and 2016. In 2013, HER2 results were evaluated using two sets of four different breast cancer specimens in five laboratories. A total of 20 immunohistochemistry and 20 CISH cases were tested. In 2014, there were 6 testing rounds, and a total of 24 specimens were analyzed, whereas in 2015 and 2016, seven testing rounds were conducted, with four additional cases (i.e. a total of 28 specimens). In 2014, 2015 and 2016, all institutions performed immunohistochemical analysis only. RESULTS: We found discrepan¬cies in HER2 immunohistochemical (IHC) results in all four surveys. IHC testing resulted in diagnostic discordance between participating centers in two (2/17) cases in 2013, two (2/24) in 2014, four (4/27) cases in 2015 and three cases (3/27) in 2016. The overall agreement among the centers was 79%, 85.5%, 83.5% and 89.4%, respectively. For CISH analyses, the results for 16 (84.2%) of 19 samples were consistent for all participants. Three results were found to be discordant, indicating a misdiagnosis rate of 15.8%. In all the discrepant cases, interinstitutional discordances were related to technical and evaluation issues. CONCLUSIONS: Our study highlights the difficulty encountered during HER2 testing using immunohistochemistry and CISH. This also emphasizes the need for rigorous quality control procedures for specimen preparation and analysis.

Markers of proliferation and cytokeratins in the differential diagnosis of jaw cysts.

We conducted a retrospective study to analyze the histologic and immunohistochemical findings in three main types of odontogenic cyst. We studied 90 archived cystic jaw lesions: 30 dentigerous cysts, 30 keratocystic odontogenic tumors, and 30 radicular cysts. The cyst types were identified on the basis of clinical, radiologic, and histopathologic findings. Immunohistochemical analyses included staining with Ki-67, p53, epidermal growth factor receptor (EGFR), cytokeratin (CK) 8, CK14, CK17, and CK18. Cell immunopositivity was evaluated for the entire epithelium. The criteria for Ki-67 and p53 positivity were dense and/or faint nuclear staining, and cells were considered EGFR-positive if they exhibited membrane staining and/or cytoplasm staining. For the cytokeratins, cells exhibiting cytoplasm staining were considered positive. Five representative fields of each lesion were selected and identified in each of the Ki-67- and p53-stained slides. We found a statistically significant difference in the ratio of Ki-67-positive cells in the entire layer between the keratocystic odontogenic tumors and both the dentigerous cysts and the radicular cysts. A statistically significant difference was observed in the ratio of p53-positive cells between the keratocystic odontogenic tumors and the radicular cysts. Cytokeratins proved to be useful in differentiating radicular cysts from other types of cystic jaw lesions because of their CK8-positive and CK17-negative immunolabeling.

Antigen unmasking induced by superheating.

This paper is dedicated to the description of superheating as a method for antigen retrieval. In our investigation, this antigen retrieval method was used on thermal plate, heating tissue sections at temperature 120° C for 90 minutes. In the research we conducted the superheating method was applied to formalin-fixed paraffin-embedded tissue sections of breast tumors. The following monoclonal antibodies were used: estrogen receptor, progesterone receptor, epithelial membrane antigen, CD34, and Ki-67. With these tested antibodies we had good staining and no loss of tissue sections during the staining process.

Primary leptomeningeal melanocytosis--a case report with an autopsy diagnosis.

INTRODUCTION: Primary melanocytosis of the leptomeninges is a rare tumor, most likely originating from the melanocytes in the leptomeninges. The average survival is only about 5 months. CASE REPORT: A 61-years-old woman presented with headache, amaurosis and hallucinations lasted for two months, and she had been treated at the Clinic for Psychiatry and Clinic for Infectious Diseases. The cerebrospinal fluid analysis showed a lower level of glucose and a higher level of proteins. Small shaded areas of basal leptomeninges and hydrocephalus were found by computed tomography and magnetic resonance imaging. The autopsy showed a dark brown mass on basal leptomeninges with blurred boundaries. No pigmented skin lesions were found. Histopathological analysis revealed a primary leptomeningeal melanocytosis. CONCLUSION: Primary leptomeningeal melanocytosis is a rare tumor, difficult to diagnose. This case is being presented for its specificity, since this diagnosis is not frequently seen in practice.

Multinucleated stromal giant cells in adenoid cystic carcinoma of the breast: a case report and literature review.

BACKGROUND: We presented an unusual case of adenoid cystic carcinoma (ACC) of the breast with multinucleated stromal giant cells (MSGCs). To the best of our knowledge, the occurrence of ACC with MSGCs has not been reported previously. MSGCs should be distinguished from other multinucleated giant cells in breast tumors. The histogenesis of MSGCs still remains obscure. In hope to elucidate the histogenesis of MSGCs, we used a broad range of antibodies. CASE REPORT: A 40-year-old woman presented with a palpable lump in the subareolar location of her right breast. Excision of the tumor was performed. At gross pathologic examination the tumor was 20 x 15 x 15 mm in size, red-brown and well circumscribed. The surgical margins were positive for carcinoma and skin-sparing mastectomy with axillary dissection was complited. Eighteen lymph nodes examined were uninvolved. Pathohistological examination showed ACC with numerous MSGCs scattered within tumor stroma. Immunohistochemical studies indicated that MSGCs are probably derived from stromal fibroblasts. These cells showed strong reactivity only for vimentin. Staining for histiocytic marker (CD68), as well as for epithelial marker (cytokeratin), myoepithelial markers (S-100, alpha-smooth muscle actin), vascular marker (CD34), hormonal receptors (ER, PR) and HER2 in MSGCs were negative. CONCLUSION: The presence of MSGCs should not alter the prognosis of an otherwise typical breast ACC.

An approach to malignant mammary phyllodes tumors detection.

BACKGROUND/AIM: Mammary phyllodes tumors (MPT) are uncommon fibroepithelial (biphasic) neoplasms whose clinical behavior is difficult to predict on the basis of histological criteria only. They are divided into benign, borderline malignant and malignant groups. Sometimes it appears difficult to distinguish these tumors from other types of soft tissue sarcomas. Because of the relatively scant data on the role of biological markers in MPT histogenesis, we have decided to undertake the following study, trying to shed more light on the issue by investigating the following elements that make up MPT: their histological patterns, biological behavior, enzymohistochemical, histochemical and immunohistochemical characteristics (ICH) together with the mast cell analysis. METHODS: We examined the biopsy material of 35 MPT in our laboratory. Enzymohistochemistry was performed on frozen sections (method of Crowford, Nachlas and Seligman). The used methods were classical hematoxylin-eosin (H & E); histochemical Massontrichrome, Alcian-blue, Periodic acid Schiff and immunohistochemical LSAB2 method (DacoCytomation). Ki-67, c-kit, vimentin, estrogen receptor (ER), progesterone receptor (PR) and Her-2 oncoprotein immunohistochemistry was performed on all tumors. RESULTS: The patients were ranged per age from 30--62 years (mean 43.3 years, median 39 years). A total of 35 cases of MPT were included: 20 benign (57%), 6 borderline malignant (17%) and 9 malignant (26%). Twenty-two patients (62.8%) underwent segmental mastectomy, while 13 (37.2%) had total mastectomies. Twenty-eight patients had negative surgical margins at original resection. The mean size of malignant MPT (7.8 cm) was larger than that of benign MPT (4.5 cm). Significant features of the malignant MPT were: stromal cellularity, stromal cellular atypism, high mitotic activity, atypic mitoses, stromal overgrowth, infiltrative tumor contour and heterologous stromal elements. Benign MPT showed strong enzymohistochemical Leucine Amino Peptidase (LAP) activity in both epithelial and stromal components while it was weak or absent in the epithelial parts of the malignant tumors. Acid mucopolysacharides were present in the stromal component of all types of these tumors. Benign MPT had a lower Ki-67 than did borderline malignant MPT (4 versus 28). Malignant MPT had a greater than 8-fold higher Ki-67 activity than did benign tumors (35 versus 4). Intracyto-plasmatic c-kit expression was associated with a pathological diagnosis of malignant MPT, correlating with increasing grade (p < 0.05). In hypercellular stroma of borderline malignant and especially malignant forms of MPT, high activity of ER in mast cells was confirmed. Oncoprotein Her-2 activity, mostly in epithelial components, correlated with the degree of malignant progression of MPT (p < 0.05). CONCLUSION: Besides the well-known malignant features additional parameters have been found to be high Ki-67 and c-kit stromal expressions, and weak LAP activity in the epithelial part of malignant MPT, as well as mast cells with a high expression of ER.

Immunohistohemical evidences of pregnancy in uterine curettage tissue by the use of a double immunocytochemical staining technique using cytokeratin 7 and vimentin antibodies.

BACKGROUND/AIM: Usual histopatological diagnosis of intrauterine pregnancy is made by demonstration of chorionic villi, but in the curettage tissue from intrauterine miscarriage they may not be present in all cases. The use of monoclonal antibody against cytokeratin as a sensitive and reliable marker for the morphologic discrimination between invasive trophoblastic (IT) cells and decidual cells has been well established. The aim of this study was to determine the presence of pregnancy in endometrial curettings when chorionic villi are absent from patients suspected of intrauterine pregnancy. METHODS: Twenty cases of endometrial tissue specimens were investigated for cytokeratin and vimentin expression by a double immunostaining for detection of IT cells. RESULTS: Out of the total number of cases (20) 17 cases expressed cytokeratin 7 positive IT cells, that are an evidence of pregnancy. CONCLUSION: The obtained results indicated, that double immunohistochemical demonstration of cytokeratin and vimentin is useful for identifying pregnancy in all chorionic villi-negative cases.