Expression of CD8, CD68 and HER2 markers in breast carcinoma in diabetes patients

Background and Aims: Breast cancer is one of the leading causes of mortality among women. The aim of our study is to evaluate the stromal expression of CD10 in invasive breast carcinoma and to find its relationship with other prognostic markers such as age, histopathologic grade, estrogen receptor (ER), progesterone receptor (PR), and HER2Neu status. Materials and Methods: A total of 51 cases of breast cancer were included in the study. Representative sections were taken and hematoxylin and eosin staining was done. Immunohistochemistry was performed with ER, PR, Her2neu, and CD10. Stromal expression of CD10 is positive when >10% of stromal cells show cytoplasmic and membranous positivity in invasive breast carcinoma. This will be noted and statistically analyzed with different known prognostic markers of breast carcinoma. Results: Stromal CD10 positivity was seen in 74.5% of cases (45% were strongly positive and 29.5% were weakly positive). 25.5% of cases were negative. Positivity for ER, PR, and HER2, was 58.8%, 35.3%, and 27.5%, respectively. Stromal expression of CD10 was found to be significantly associated with ER negativity (P = 0.001) and PR negativity (P = 0.001). Increasing intensity was noted with few parameters such as postmenopausal status and histologic grade but could not show statistical significance. No correlation was found between CD10 overexpression with respect to the age, tumor size, lymph node positivity status, tumor stage and HER2Neu status. Conclusions: CD10 expression correlated strongly with well-established negative prognostic markers, ER/PR negativity, and higher tumor grade, thus indicating that CD10 can be used as an independent marker indicating poor prognosis. This study highlights the role of stromal CD10 expression in predicting prognosis and the relationship with other prognostic markers. Keeping the role stroma plays in predicting prognosis and tumor response, CD10 can be included as a routine prechemotherapy marker in breast carcinoma. Further studies should be performed to see the role stroma plays in hormonal expression and the usefulness of CD10 to predict treatment failure in breast carcinomas receiving neoadjuvant therapy.

Background and aim Breast cancer (BC) is the second most common global cause of cancer deaths among women. Several immune cells are identified in the tumor microenvironment of BC patients, including tumor‐associated macrophages. We aimed at exploring the expression of distinct functional phenotypes using macrophages’ markers, where CD68 is a pan‐macrophage marker, CD86 is a marker expressed in polarized M1 subtype, and CD163 is expressed in M2 polarized subtype. Methods A retrospective study was performed using 90 formalin‐fixed paraffin‐embedded BC specimens for the immunohistochemical analysis of CD68, CD86 and CD163. Also, an in silico tool, UALCAN, was used on a larger cohort (n=1,081) of BC patients to investigate the expression of these markers. The macrophages’ markers were then associated with the clinicopathological parameters of BC patients. Triple‐negative BC cell line “CAL‐51” and luminal BC cell line “MCF‐7” were used to collect their respective supernatants that were added to THP‐1 derived macrophages. Then CD86 and CD163 were assessed using western blot. Results The pan‐marker of macrophages, CD68, along with the M1 CD86 marker and M2 CD163 marker, showed positive results in all the 90 investigated patients (Figure 1). CD86 expression was significantly associated with body mass index (BMI) and Ki‐67 proliferation marker. On the other hand, CD163 was significantly correlated with tumor size, estrogen, progesterone receptors, and BC molecular subtypes. Moreover, the high expression of CD86 and CD163 showed unfavorable outcome and survival of BC patients. In silico analysis revealed a significant increase in the CD86 expression in BC patients, especially in the triple‐negative subtype. Similarly, CD163 expression was found to be higher in the triple‐negative subgroup compared to the luminal group (Figure 2A). Additionally, in vitro work showed that the macrophages induced from the monocytic THP‐1 cell line express high levels of CD163 upon the exposure to conditioned media collected from the luminal BC cell line “MCF‐7”, thus showing M2 phenotype. On the contrary, CD163 expression was reduced upon the exposure to the conditioned media collected from the triple‐negative BC cell line “CAL51”, indicating more M1 phenotype (Figure 2B). Conclusion Tumor‐associated macrophages are associated with cancer progression and molecular subtypes. Hence, identifying the macrophages polarization profiles in each molecular subtype might aid their use as potential therapeutic targets.

Wnt9b was recently identified as a highly sensitive and specific marker for breast carcinomas. Due to the limited number of triple-negative breast carcinomas (TNBCs) in previous study, we further explored Wnt9b's utility in breast carcinoma, especially in TNBCs including metaplastic carcinomas. We systematically evaluated Wnt9b expression on tissue microarrays (TMAs) from 413 breast carcinomas, 208 urothelial carcinomas, 102 endometrial carcinomas, 109 cholangiocarcinomas, 192 ovarian carcinomas, 48 lung adenocarcinomas, 69 colorectal adenocarcinomas, and 78 melanomas, and whole tissue section (WTS) from 20 human epidermal growth factor receptor 2-positive, 34 nonmetaplastic TNBCs, and 67 invasive metaplastic carcinomas. The results showed Wnt9b was highly expressed in breast carcinomas (91% on TMA and 98% on WTS) and in nonmetaplastic TNBCs (91% on TMA and 97% on WTS), but almost completely negative in other tested tumor types. Wnt9b was also highly expressed in metaplastic carcinomas (80%), significantly higher than GATA3 (56%) and SOX10 (48%), but slightly lower than TRPS1 (90%). In summary, our results demonstrate that Wnt9b is a highly sensitive marker for breast carcinomas, including TNBCs and metaplastic carcinomas. Further, we compared its utility with other breast markers including TRPS1, GATA3, and SOX10 in metaplastic carcinomas.

Utility of TRPS-1 immunohistochemistry in diagnosis of metastatic breast carcinoma in cytology specimens

SMAD4 is a downstream mediator of transforming growth factor beta. While its tumor suppressor function has been investigated as a prognostic biomarker in several human malignancies, its role as a prognostic marker in breast carcinoma is still undefined. We investigated SMAD4 expression in breast carcinoma samples of different histologic grades to evaluate the association between SMAD4 and outcome in breast cancer. We also investigated the role of SMAD4 expression status in MDA-MB-468 breast cancer cells in responding to TGF-β stimulation. SMAD4 expression was assessed in 53 breast ductal carcinoma samples and in the surrounding normal tissue from 50 of the samples using immunohistochemistry, Western blot, and real-time PCR. TGF-β-SMAD and non-SMAD signaling was assessed by Western blot in MDA-MB-468 cells with and without SMAD4 restoration. SMAD4 expression was reduced in ductal breast carcinoma as compared to surrounding uninvolved ductal breast epithelia (p < 0.05). SMAD4 expression levels decreased from Grade 1 to Grade 3 ductal breast carcinoma as assessed by immunohistochemistry (p < 0.05). Results were recapitulated by tissue array. In addition, immunohistochemistry results were further confirmed at the protein and mRNA level. We then found that non-SMAD MEK/MAPK signaling was significantly different between SMAD4 expressing MDA-MB-468 cells and SMAD4-null MDA-MB-468 cells. This is the first study indicating that SMAD4 plays a key role in shifting MAPK signaling. Further, we have demonstrated that SMAD4 has a potential role in the development of breast carcinoma and SMAD4 was a potential prognostic marker of breast carcinoma. Our findings further support the role of SMAD4 in breast carcinoma development. In addition, we observed an inverse relationship between SMAD4 levels and breast carcinoma histological grade. Our finding indicated that SMAD4 expression level in breast cancer cells played a role in responding non-SMAD signaling but not the canonic SMAD signaling. Further mechanistic studies are necessary to establish the role of SMAD4 in breast carcinoma prognosis and potential specific targeting.

Paired-like homeodomain transcription factor 2 (PITX2) is another new marker in breast carcinoma since hypermethylation at P2 promoter of this gene was noted to be associated with poor prognosis. We investigated the expression of PITX2 protein using immunohistochemistry in invasive ductal carcinoma and its association with the established growth receptors such as estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth receptor 2 (HER2). We conducted a cross sectional study using 100 samples of archived formalin-fixed paraffin embedded tissue blocks of invasive ductal carcinoma and stained them with immunohistochemistry for PITX2, ER, PR and HER2. All HER2 with scoring of 2+ were confirmed with chromogenic in-situ hybridization (CISH). PITX2 protein was expressed in 53% of invasive ductal carcinoma and lack of PITX2 expression in 47%. Univariate analysis revealed a significant association between PITX2 expression with PR (p=0.001), ER (p=0.006), gland formation (p=0.044) and marginal association with molecular subtypes of breast carcinoma (p=0.051). Combined ER and PR expression with PITX2 was also significantly associated (p=0.003) especially in double positive cases. Multivariate analysis showed the most significant association between PITX2 and PR (RR 4.105, 95% CI 1.765-9.547, p=0.001). PITX2 is another potential prognostic marker in breast carcinoma adding significant information to established prognostic factors of ER and PR. The expression of PITX2 together with PR may carry a very good prognosis.

CD47 expression and tumor-associated immune cells in breast cancer and their correlation with molecular subtypes and prognostic factors.

Breast cancer is one of the leading causes of mortality in Indian women. Although breast cancer is an epithelial malignancy, stroma plays a key role in its development and pathogenesis. Stromal markers are now emerging as novel markers in assessing the prognosis of invasive breast cancer and have not been studied extensively till date. The aim of the present study is to study the stromal expression of CD10 in breast carcinoma, find its relationship with other prognostic markers and study the role stroma plays in breast cancer pathogenesis. A total of 70 cases of breast cancer were included in the study. Representative sections were taken and hematoxylin and eosin staining was done. Immunohistochemistry was performed with ER, PR, Her2neu and CD10. Stromal expression of CD10 (>10% stromal positivity was considered positive) in invasive breast carcinoma was noted and was statistically analyzed with different known prognostic markers of breast carcinoma. Stromal expression of CD10 was found to be significantly associated with increasing tumor grade (P = 0.04), increasing mitotic rate (P = 0.33), worsening prognosis (P = 0.01), ER negativity (P = 0.0001), Her2neu positivity (P = 0.19) and with molecular subtypes (CD10 positivity with the HER2 type, and CD10 negativity with Luminal type). No correlation was found between CD10 overexpression and PR, age, menopausal status, tumor size, lymph node positivity and tumor stage. This study gives substantial proof to the various models/research papers explaining the role of stroma/CD10 in breast cancer pathogenesis. Keeping the role stroma plays in predicting prognosis and tumor response, CD10 should be included as a routine pre-chemotherapy marker in breast carcinoma. Further studies should be performed to see the role stroma plays in hormonal expression and the usefulness of CD10 to predict treatment failure in breast carcinomas receiving neoadjuvant therapy.

Expression of novel neuroendocrine markers in breast carcinomas: a study of INSM1, ASCL1, and POU2F3

Lipopolysaccharide (LPS)-tolerant monocytes produce small amounts of inflammatory cytokines, which is one of the characteristics of the alternative activated macrophages (AAM). These cells exhibited an increased expression of CD206 and CD163. Given the functional similarities of AAMs with the modulation of monocytes' functions observed during sepsis and LPS-tolerance, we evaluated whether the inhibition of inflammatory cytokine production by LPS-tolerant monocytes is associated with the phenotype of cells expressing CD206 and CD163. We investigated whether tolerant human monocytes would modulate their expression of CD206 and CD163, markers of alternative activation, and whether the level of their expression would be related to cytokines detection. Tolerance to LPS was induced in peripheral blood mononuclear cell by pre-incubating the cells with increasing concentrations of LPS. The expression of CD206 and CD163 and intracellular TNF-α and IL-6 was determined 24 h after LPS challenge by flow cytometry. No differences in CD163 expression were observed between tolerant and non-tolerant cells, while the expression of CD206, which was decreased following LPS stimulation in non-tolerized cells, was further reduced in tolerant cells. Decreased production of inflammatory cytokines was observed in the tolerized cells, regardless of the expression of CD163 and CD206, with the exception of IL-6 in CD206+ monocytes, which was similarly expressed in both tolerized and non-tolerized cells. The effect of LPS in the expression of CD163 and CD206 on monocytes is not reverted in LPS tolerant cells, and the inhibition of inflammatory cytokines in tolerant cells is not related with modulation of these receptors. © 2016 International Clinical Cytometry Society.

Study question We aim to determine the percentages of CD25-positive and CD69-positive lymphocytes in peripheral blood of endometriosis (EMS) patients. Summary answer Higher percentages of lymphocytes expressing CD69 and CD25 markers in EMS and their correlation with the severity of the disease, indicate persistent activation of lymphocytes What is known already Exposure to antigens causes activation markers to appear on the surface of lymphocytes. Among them, we can distinguish early and late activation markers. The earliest one is CD69, which is expressed upon activation by the TCR receptor. CD69 plays a role in the proliferation and survival of activated T lymphocytes. CD25 is a moderate late activation marker, considered the most important marker of cellular response activation. The significance of CD69 and CD25 expression by T cells in endometriosis has not yet been determined. Increased CD69 expression on various lymphocyte subsets in peritoneal fluid in EMS has been reported. Study design, size, duration Within this prospective study between January 2016 and August 2018 immune diagnostics of the number of CD25-positive and CD69-positive lymphocytes in peripheral blood of EMS patients (firstly diagnosed during laparoscopy and confirmed histopatologically) and non-EMS controls were performed using flow cytometry analysis. Total number of 74 subjects were included in the study. All patients signed written informed consent before their enrollment in the study. Participants/materials, setting, methods We enrolled 54 subjects with previously untreated endometriosis and 20 healthy age-matched controls. Peripheral blood was collected from control patients to assess the immunophenotype of lymphocytes and measure expression of activation markers Cd25 and CD69 on several subtypes B and T lymphocytes. Diagnosis and assessment of the stage of the EMS was established during laparoscopy using rASRM score. Differences were considered statistically significant with a p &amp;lt; 0.05. Main results and the role of chance Significantly higher expression of the CD25 and CD69 antigen was found in both CD3+ T, CD4+ T and CD8+ T cells as well as CD19+ B cells. In each of the studied groups, a significance level of p &amp;lt; 0.001 was obtained. There was a weak positive correlation between the percentage of CD4+CD25+ T cells and the stage of endometriosis (R = 0.357; p = 0.008) and negative correlations between the percentage of endometriosis and the percentage of CD3+CD69+ T cells (R = −0.554; p &amp;lt; 0.001), T CD4+CD69 + (R = −0.554, p &amp;lt; 0.001) and T CD8+CD69 + (R = −0.553, p &amp;lt; 0.001). The expression of activation markers, CD25 and CD69 antigens in patients suffering from endometriosis with accompanying clinical symptoms was also evaluated. n the group of patients with endometriosis accompanied by infertility or pelvic pain, no statistical differences in the expression of CD25 and CD69 antigens were observed. The only clinical condition coexisting with statistically different expression of activation markers is adhesion disease accompanying endometriosis - a statistically significantly lower percentages of T CD3+CD69 + (p = 0.002), T CD4+CD69 + (p = 0.002) and T CD8+CD69 + (p = 0.002). Limitations, reasons for caution Small sample size is an important limitation of this study. In addition, evaluating immunological analysis only in serum samples does not let us drive any conclusions on the local changes of endometriosis lesions. Wider implications of the findings Clinical interpretation of changed expression of CD69 and CD25 in EMS could be useful in evaluating mechanisms of cellular activation, peripheral tolerance and immune imbalance involved in pathogenesis of endometriosis. Further studies to understand mechanisms underlying correlation between stage of the disease and expression of CD69 should be considered. Trial registration number not applicable

Introduction/Objective Gene expression profiling of human tumors has introduced a new approach for classifying breast cancers. Estrogen receptor (ER), progesterone receptor (PR), and HER-2/neu (HER2) status provides crucial prognostic and predictive information for these patients. A project was designed to find the overall proportion of prognostic markers in breast carcinoma in our tertiary institution. Methods/Case Report In a retrospective study, breast specimens between January 1, 2024, and December 31, 2024, were evaluated. 233 specimens (132 biopsies, 101 lumpectomies/ mastectomies) had a diagnosis of invasive ductal carcinomas (n = 157), DCIS (n = 46), LCIS (n = 4), invasive lobular carcinomas (n = 15), metaplastic carcinomas (n = 6), mucinous carcinomas (n = 3) and pure tubular carcinomas (n = 2). They all had subsequent ER, PR, Her-2/Neu testing Results The overall proportion of ER-negative breast cancers (n = 31) was 13.3%. The majority of invasive carcinomas (79.6%, n = 137) had ER &amp;gt; 60% positivity. Out of all invasive carcinomas, 16.8% (n = 29) were triple negative. These were all invasive ductal grade 2-3/3 or metaplastic carcinoma. The overall proportion of HER2-positive breast cancers, tested by IHC or FISH, was 10.7% (n = 25). Conclusion Our findings correlate with the CAP benchmark which establishes that the proportion of ER-negative breast cancers should not exceed 30%, and that the overall proportion of HER2-positive breast cancers is 10 to 25%.

To analyse the diagnostic role of trefoil factor-1 and -3 (TFF1, TFF3), forkhead box protein A1 (FOXA1), carbonic anhydrase XII (CA XII) and trichorhinophalangeal syndrome type 1 (TRPS1) in serous effusions. The prognostic role of these markers in breast carcinoma was additionally studied. Protein expression by immunohistochemistry was analysed in 247 effusions, consisting of 60 breast carcinomas, 54 tubo-ovarian carcinomas, 47 mesotheliomas, 44 lung carcinomas, 20 uterine corpus and cervical carcinomas, 17 gastrointestinal carcinomas and 5 cancers of other origin. TFF1, TFF3, FOXA1, CA XII and TRPS1 expression was found in 67%, 70%, 88%, 82% and 83% of breast carcinomas, respectively. Expression of all markers was seen in some carcinomas of other origin, most commonly in GI metastases, but was least frequent for TRPS1. CA XII expression was additionally seen in mesotheliomas and reactive mesothelial cells. All 5 markers were significantly overexpressed in breast compared to tubo-ovarian carcinoma (all p < 0.001) and lung carcinoma (all p < 0.001 except for FOXA1, p = 0.023). TFF1 (p = 0.003), TFF3 (p = 0.008) and FOXA1 (p = 0.017) expression was significantly higher in receptor-positive compared to receptor-negative primary breast carcinomas. In survival analysis for 44 breast carcinoma patients with clinical data, TFF1 expression was associated with a trend for longer overall (p = 0.096) and disease-free (p = 0.06) survival. TFF1, TFF3, FOXA1, CA XII and TRPS1 are sensitive breast carcinoma markers, with FOXA1 performing best in terms of sensitivity and TRPS1 being the most specific. Whether the expression of these markers in breast carcinoma effusions is informative of survival merits further research.

BackgroundSepsis causes a high rate of mortality and long-term morbidity, associated with an imbalance of innate immunity against infections and inflammation. Obesity and diabetes increase the risk for disease severity. Monocyte dysfunction plays a major role and justify further investigations.ObjectiveTo investigate the distribution and inflammatory phenotypes in circulating monocyte subsets in patients manifesting with sepsis including septic shock with and without obesity and diabetes.MethodsA total of 235 blood samples were tested from critically ill adult patients registered at the intensive care unit (ICU). The cohorts were divided into non-diabetic groups with or without obesity and diabetic groups with or without obesity, suffering from sepsis or septic shock. We determined frequencies of total monocytes and of monocyte subsets in the circulation and density expression levels of functional markers, including CD14, CD16, HLA-DR, CD33, CD163, CD206, and arginase-1 by flow cytometric analysis.ResultsWhen progressing to septic shock in non-diabetic and diabetic patients, the percentages of total monocytes among the leukocyte population and of CD33+ and CD14+ monocytes among the monocyte population were consistently down-regulated compared to non-sepsis in non-diabetic and diabetic patients, respectively. Non-diabetic sepsis patients further presented with decreased CD33 and up-regulated CD163 expression density, which was absent in diabetic patients. We subsequently addressed obesity-related changes of monocytes in non-diabetic and diabetic septic patients. Obese septic patients with diabetes were unique in displaying increased monocytic CD16 and CD163 expression. However, obese septic patients without diabetes solely presented with lower amounts of non-classical monocytes. Body mass index (BMI) dependent changes were restricted to diabetic septic patients, with a significantly higher diminution of the classical monocyte subset and concomitantly increased CD16 expression densities.ConclusionDistribution and phenotypes of monocyte subsets were differentially modulated in critically ill patients with and without metabolic disease when progressing to sepsis or septic shock. Only diabetic septic patients displayed decline of classical monocytes and increase of CD16 expression densities. Therefore, diabetes but not obesity appears to promote the inflammatory phenotype of circulating monocytes in critically ill patients.

Utilization of the GCDFP-15 Protein as a Marker of Breast Carcinomas with Apocrine Features: Clinical and Pathological Findings