Proliferative Activity of Myoepithelial Cells in of Mucoepidermoid Carcinoma

AIM: The aim of the study is to investigate the role of myoepithelial cells in the pathogenesis of mucoepidermoid carcinoma (MEC) using the double immunohistochemical staining; α _smooth muscle actin (_α-SMA)as specific marker for the myoepithelial cell differentiation and proliferative cell nuclear antigen (PCNA) as a marker for proliferative activity of myoepithelial cells. MATERIAL AND METHODS: Retrospective study of twenty salivary gland specimens (ten MEC and ten normal salivary glands) were studied using double immunohistochemical labeling for α _smooth muscle actin α-SMA) and proliferative cell nuclear antigen (PCNA). The SPSS statistical package was used for data analysis (IBM SPSS Statistics for Windows, Version 20.0, Released 2011, IBM Corp, and Armonk, NY, USA). RESULTS: In mucoepidermoid carcinomas, no positivity of α-SMA was seen in neoplastic cells (Frequent test), and it was just observed in the stroma of tumor, in the walls of blood vessels whereas, PCNA was positive, especially in high-grade tumors. In contrast, in normal salivary glands, the proliferating myoepithelial cells are stained by both α-SMA and PCNA. CONCLUSIONS: We believe that the myoepithelial cells have no a role in the development of mucoepidermoid carcinoma.

Expression of Components of the Renin-Angiotensin System by Cancer Stem Cells in Renal Clear Cell Carcinoma

This study investigated the expression of components of the renin-angiotensin system (RAS) by cancer stem cells (CSCs) we have recently demonstrated in renal clear cell carcinoma (RCCC). Fifteen RCCC tissue samples underwent immunohistochemical staining for components of the RAS: renin, pro-renin receptor (PRR), angiotensin-converting enzyme (ACE), angiotensin-converting enzyme 2 (ACE2), and angiotensin II receptor 2 (AT2R). Immunofluorescence co-staining or double immunohistochemical staining of these components of the RAS with stemness-associated markers OCT4 or KLF4 was performed on two of the samples. Protein and transcript expression of these components of the RAS in six RCCC tissue samples was investigated using western blotting and reverse transcription quantitative polymerase chain reaction (RT-qPCR), respectively. In addition, angiotensin II receptor 1 (AT1R) was investigated using RT-qPCR only. Immunohistochemical staining demonstrated expression of renin, PRR, and ACE2 in 11, 13, and 13 out of 15 RCCC samples, respectively, while AT2R was expressed in all 15 samples. ACE was detected in the endothelium of normal vasculature only. Double immunohistochemical staining demonstrated localization of ACE2, but not renin, to the KLF4+ CSCs. Immunofluorescence staining showed localization of PRR and AT2R to the OCT4+ CSCs. Western blotting confirmed protein expression of all components of the RAS except renin. RT-qPCR demonstrated transcript expression of all components of the RAS including AT1R, but not AT2R, in all six RCCC tissue samples. This study demonstrated expression of PRR, ACE2, and AT2R by the CSCs within RCCC. Further studies may lead to novel therapeutic targeting of CSCs by manipulation of the RAS in the treatment of this aggressive cancer.

Gastric Carcinomas with Stromal B7-H3 Expression Have Lower Intratumoural CD8+ T Cell Density

CD8+ T cells are the main effector cells of anti-cancer immune response that can be regulated by various costimulatory and coinhibitory molecules, including members of the B7 family. B7 homolog 3 (B7-H3) appears as a promising marker for immunotherapy; however, its significance in gastric cancer (GC) is unclear yet. We evaluated the spatial distribution of CD8+ T cells in relation to the expression of B7-H3 by double immunohistochemical staining. The level of B7-H3 intensity was scored manually (0–3) and dichotomized into B7-H3-low and B7-H3-high groups. The distribution and density of CD8+ T cells was analysed using whole slide digital imaging. B7-H3 was expressed mainly in the stromal compartment of GC (n = 73, 76% of all cases). Tumours with high expression of B7-H3 showed larger spatial differences of CD8+ T cells (86.4/mm2 in tumour centre vs. 414.9/mm2 in invasive front) when compared to B7-H3-low group (157.7/mm2 vs. 218.7/mm2, respectively) (p < 0.001). This study provides insight into the expression pattern of B7-H3 in GC of Western origin. In GCs with higher level of B7-H3 expression, CD8+ T cells were spatially suppressed in the tumour centre suggesting that B7-H3 might be involved in tumour escape mechanisms from the immune response.

Reevaluating Significance of Perineural Invasion in Gastric Cancer Based on Double Immunohistochemical Staining

Context.—In gastric cancer, the significance of perineural invasion remains controversial. Detecting perineural invasion with hematoxylin-eosin staining often leads to misdiagnosis. Labeling nerves by immunohistochemistry greatly assists perineural invasion detection, but it might also be misdiagnosed, because scattered cancer cells are difficult to recognize. Objective.—To reevaluate the significance of perineural invasion in gastric cancer by double immunohistochemical staining that labels both nerves and cancer cells, and to examine agreements on perineural invasion detection between double immunohistochemical staining and single immunochemical staining (to label nerves) or hematoxylin-eosin staining. Design.—We evaluated perineural invasion in 160 cases of gastric cancer with double immunohistochemical staining, single immunochemical staining, and hematoxylin-eosin staining, respectively; then we investigated the prognostic significance of perineural invasion. Results.—Perineural invasion was detected in 65.0% (104 of 160), 38.1% (61 of 160), and 56.9% (91 of 160) of cases with double immunohistochemical staining, hematoxylin-eosin staining, and single immunohistochemical staining, respectively. Agreement was low between double staining and hematoxylin-eosin staining (κ = .34), and most false reports occurred in diffuse gastric cancer. Agreement between single immunochemical staining and double staining was good (κ = .67), but it declined in diffuse gastric cancer (κ = .28). Perineural invasion was closely associated with other clinicopathologic variables. Although perineural invasion–positive patients had a worse outcome than perineural invasion–negative patients, it was not an independent prognostic factor (P = .11; hazard ratio, 0.637; 95% confidence interval, 0.366–1.110). Conclusions.—Double immunohistochemical staining could improve accuracy of perineural invasion detection in gastric cancer, particularly in the diffuse type. Moreover, perineural invasion predicts a poor outcome in gastric cancer, but it cannot provide more information than traditional clinicopathologic variables.

Strong Correlation Between Angiogenesis and Macrophage Counts in Waldenstrom’s Macroglobulinemia: Implications into the Biology of the Disease

Angiogenesis represents an essential step of disease progression in several hematological malignancies. A Mayo Clinic study reported that microvessel density (MVD) was increased (intermediate- or high- grade angiogenesis) in 30% of patients with Waldenstrom s Macroglobulinemia (WM), showed only weak correlation with marrow infiltration and had no impact on patients’ survival [Rajkumar et al, Semin Oncol2003;30:262-4]. Macrophage inflammatory protein-1 alpha (MIP-1alpha) is a potent chemoattractant for macrophages, which contributes to increased angiogenesis in malignant diseases, including multiple myeloma. Our group has reported that serum levels of MIP-1alpha are elevated in WM. To further elucidate the role of angiogenesis in WM, we investigated the association between MVD, MIP-1alpha expression and the macrophage numbers in trephine biopsies of 34 patients with newly-diagnosed WM (3 with asymptomatic disease) and 3 with IgM-Monoclonal Gammopathy of Undetermined Significance (MGUS). Bone marrow biopsies were studied using double immunohistochemical staining for CD34 (endothelial cells) and CD68 (macrophages/mast cells) using antibodies from Becton Dickinson, San Jose, CA, USA & Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, respectively. We have also used double immunohistochemical staining for CD20/MIP-1alpha and for CD138/MIP-1alpha using an anti-MIP-1alpha antibody from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA) to evaluate the MIP-1alpha expression by WM cells. Thirteen patients (35%) showed intermediate-grade and 4 (10%) high-grade angiogenesis. All patients with IgM-MGUS and asymptomatic WM had a very low microvessel count (median: 1, range: 1–2), while the median microvessel count for symptomatic WM was 4 (range: 1–8, p&lt;0.01). There was a strong correlation between the grade of angiogenesis (as assessed by the microvessel counts) and the number of macrophages into the “hot-spots” (r=0.823, p&lt;0.0001). Furthermore, statistically significant correlations were observed between the percentage of lymphoplasmacytoid cell infiltration of the bone marrow with microvessel counts (r=0.554, p=0.002) and macrophage numbers into the “hot spots” (r=0.457, p=0.011). WM patients with intermediate or high grade angiogenesis had increased IgM levels (p=0.007), lower hemoglobin levels (p=0.024), and reduced platelet counts (p=0.043), while patients with high-grade angiogenesis had a tendency to higher incidence of lymphadenopathy compared with all others (3/4, 75% vs. 9/33, 27%; p=0.054). There was no correlation between angiogenesis and survival. We have also observed that WM cells of all patients produced MIP-1alpha. CD138 positive WM cells had higher expression of MIP-1alpha compared to CD20 positive WM cells (p=0.001). Patients with increased numbers of CD68 positive macrophages had increased expression of MIP-1alpha by their WM cells (r=0.732, p&lt;0.001). The results of our on going study suggest that WM cells produce MIP-1alpha to attract macrophages in their bone marrow microenvironment. These macrophages seem to play a significant role in the angiogenesis process and are possibly implicated into the biology of WM.