Prognostic Value of "Basal-like" Morphology, Tumor-Infiltrating Lymphocytes and Multi-MAGE-A Expression in Triple-Negative Breast Cancer.

"Basal-like" (BL) morphology and the expression of cancer testis antigens (CTA) in breast cancer still have unclear prognostic significance. The aim of our research was to explore correlations of the morphological characteristics and tumor microenvironment in triple-negative breast carcinomas (TNBCs) with multi-MAGE-A CTA expression and to determine their prognostic significance. Clinical records of breast cancer patients who underwent surgery between January 2017 and December 2018 in four major Croatian clinical centers were analyzed. A total of 97 non-metastatic TNBCs with available tissue samples and treatment information were identified. Cancer tissue sections were additionally stained with programmed death-ligand 1 (PD-L1) Ventana (SP142) and multi-MAGE-A (mAb 57B). BL morphology was detected in 47 (49%) TNBCs and was associated with a higher Ki-67 proliferation index and histologic grade. Expression of multi-MAGE-A was observed in 77 (79%) TNBCs and was significantly associated with BL morphology. Lymphocyte-predominant breast cancer (LPBC) status was detected in 11 cases (11.3%) and significantly correlated with the Ki-67 proliferation index, increased number of intratumoral lymphocytes (itTIL), and PD-L1 expression. No impact of BL morphology, multi-MAGE-A expression, histologic type, or LPBC status on disease-free survival was observed. Our data suggest that tumor morphology could help identify patients with potential benefits from CTA-targeting immunotherapy.

Alteration of Collagen Content and Macrophage Distribution in White Adipose Tissue under the Influence of Maternal and Postnatal Diet in Male Rat Offspring.

Background and Objectives: The extracellular matrix is important for adipose tissue growth, and numerous interactions between adipocytes and extracellular matrix components occur during adipose tissue development. The main objective of this study was to investigate the interaction and influence of maternal and postnatal diet on adipose tissue remodeling in Sprague Dawley offspring. Materials and Methods: 10 Sprague Dawley females were randomly divided into two groups at nine weeks of age and fed a standard laboratory diet or high-fat diet for six weeks. Then, they were mated, and after birth, their male rat offspring were divided into four subgroups according to diet. After euthanizing the offspring at 22 weeks of age, samples of subcutaneous, perirenal and epididymal adipose tissue were collected. Sections were stained with Mallory's trichrome and analyzed by immunohistochemistry for CD68+ and CD163+ cells. Results: Staining of extracellular components showed higher collagen deposition in the perirenal and epididymal depot of offspring fed a high-fat diet. The number of CD163/CD68+ cells in the perirenal adipose tissue was lower in the CD-HFD group compared with other groups, and in the subcutaneous fat pad when the groups with modified diet were compared with those on non-modified diet. Conclusion: Morphological changes in adipose tissue, increased collagen deposition, and changes in macrophage polarization may be related to intergenerational changes in diet.

Prognostic Significance of Lymphocyte Infiltrate Localization in Triple-Negative Breast Cancer.

High infiltration by tumor-infiltrating lymphocytes (TILs) is associated with favorable prognosis in different tumor types, but the clinical significance of their spatial localization within the tumor microenvironment is debated. To address this issue, we evaluated the accumulation of intratumoral TILs (itTILs) and stromal TILs (sTILs) in samples from 97 patients with early triple-negative breast cancer (TNBC) in the center (sTIL central) and periphery (sTIL peripheral) of tumor tissues. Moreover, the presence of primary and secondary lymphoid aggregates (LAs) and the expression levels of the cancer testis antigen (CTA), NY-ESO-1, and PD-L1 were explored. High infiltration by itTILs was observed in 12/97 samples (12.3%), unrelated to age, Ki67 expression, tumor size, histologic type and grade, and LA presence. NY-ESO-1 was expressed in tumor cells in 37 samples (38%), with a trend suggesting a correlation with itTIL infiltration (p = 0.0531). PD-L1 expression was detected in immune cells in 47 samples (49%) and was correlated with histologic grade, sTILs, and LA formation. The presence of primary LAs was significantly correlated with better disease-free survival (DFS) (p = 0.027). Moreover, no tumor progression was observed during >40 months of clinical follow up in the 12 patients with high itTILs or in the 14 patients with secondary LAs. Thus, careful evaluation of lymphoid infiltrate intratumoral localization might provide important prognostic information.

COVID-19 Pandemic Effects on Breast Cancer Diagnosis in Croatia: A Population- and Registry-Based Study.

BACKGROUND: Our objective was to assess the effects of COVID-19 antiepidemic measures and subsequent changes in the function of the health care system on the number of newly diagnosed breast cancers in the Republic of Croatia. SUBJECTS, MATERIALS, AND METHODS: We performed a retrospective, population- and registry-based study during 2020. The comparator was the number of patients newly diagnosed with breast cancer during 2017, 2018, and 2019. The outcome was the change in number of newly diagnosed breast cancer cases. RESULTS: The average monthly percent change after the initial lockdown measures were introduced was -11.0% (95% confidence interval - 22.0% to 1.5%), resulting in a 24% reduction of the newly diagnosed breast cancer cases in Croatia during April, May, and June compared with the same period of 2019. However, during 2020, only 1% fewer new cases were detected than in 2019, or 6% fewer than what would be expected based on the linear trend during 2017-2019. CONCLUSION: It seems that national health care system measures for controlling the spread of COVID-19 had a detrimental effect on the number of newly diagnosed breast cancer cases in Croatia during the first lockdown. As it is not plausible to expect an epidemiological change to occur at the same time, this may result in later diagnosis, later initiation of treatment, and less favorable outcomes in the future. However, the effect weakened after the first lockdown and COVID-19 control measures were relaxed, and it has not reoccurred during the second COVID-19 wave. Although the COVID-19 lockdown affected the number of newly diagnosed breast cancers, the oncology health care system has shown resilience and compensated for these effects by the end of 2020. IMPLICATIONS FOR PRACTICE: It is possible to compensate for the adverse effects of COVID-19 pandemic control measures on breast cancer diagnosis relatively promptly, and it is of crucial importance to do it as soon as possible. Moreover, as shown by this study's results on the number of newly diagnosed breast cancer cases during the second wave of the pandemic, these adverse effects are preventable to a non-negligible extent.

Intestinal Morphology in Broiler Chickens Supplemented with Propolis and Bee Pollen.

The aim of this study was to determine the influence of dietary supplementation with propolis and bee pollen on the intestinal morphology and absorptive surface areas of chickens. Two hundred day-old Ross 308 chickens (100 male and 100 female) were equally allocated into five groups. Throughout the whole study, the control group of chickens was fed with a basal diet, while the experimental groups of chickens were fed with the same diet supplemented with propolis and bee pollen: P1 = 0.25 g of propolis/kg + 20 g of bee pollen/kg; P2 = 0.5 g of propolis/kg; P3 = 1.0 g of propolis/kg; P4 = 20 g of bee pollen/kg. The duodenal villi of chickens from all experimental groups were significantly higher and wider (p < 0.001), while their duodenal villi crypts were significantly deeper (p < 0.001) in comparison with these parameters in chickens from the control group. The villus height to crypt depth ratio, as well as the absorptive surface areas of broiler chickens, were significantly increased (p < 0.001) in experimental groups of chickens in comparison with the control group. These findings suggest that dietary supplementation with propolis and bee pollen has a beneficial effect on broilers chickens' intestinal morphophysiology.

Inter-laboratory comparison of Ki-67 proliferating index detected by visual assessment and automated digital image analysis.

BACKGROUND: Proliferation rate is a major determinant of the biologic behavior of the tumor and provides information that can be used to guide treatment decisions. METHODS: This ring study included 27 pathologists from 14 Institutions, in order to assess inter-observer concordance between pathologists in Croatia. We analyzed Ki-67 proliferative index on ten randomly selected breast cancer samples comparing consistency between visual assessment using light microscopy compared to digital image analyses results from one central laboratory as a referral value. RESULTS: When we analyzed Ki-67 as numeric value high concordance rate was found between Ki-67 score visually assessed in all participating Institutions compared to referral value assessed by digital image analysis (ICC 0.76, 95% CI 0.58-0.91), and Krippendorff's alpha was 0.79 (95% CI 0.58-1.00). Concordance was better in slides with higher Ki-67 values. When we categorized Ki-67 values according to generally accepted 20% cut-off value we noticed the lower concordance rate among participants in our study. CONCLUSION: Proliferation remains one of the most important parameters for tumor characterization helpful in making clinical decisions, but it should be used with great caution. Standardization of the Ki-67 assessment is essential and proliferating index should be expressed as exact numeric value. For patients with proliferative index near the cut-off value, other factors must be considered in making clinical decisions.

Influence of Dietary Supplementation of Propolis and Bee Pollen on Liver Pathology in Broiler Chickens.

One of the major problems in intensive breeding of chickens is liver damage. The objective of this study was to determine the influence of dietary supplementation with propolis and bee pollen on liver pathology in broiler chickens. The study was conducted on 200 Ross 308 chickens equally distributed by sex that were divided into five groups. Throughout the whole study, the control group of chickens was fed with a basal diet, while the experimental groups of chickens were fed with the same diet further supplemented with propolis and bee pollen, each supplement given separately or in combination in a certain proportion. The study showed that the clusters of lymphocytes in the hepatocytes, the vacuolar degeneration and necrosis of the liver parenchyma, the bile ductule hyperplasia, and the various forms of pathological changes in the liver arteries and veins were more frequent in liver tissue samples of the control group compared to liver tissue samples of all the experimental groups (p < 0.001). The study further showed that all the previously mentioned histopathological lesions of liver tissue were always more extensive in the liver tissue samples of the control group than in the liver tissue samples of all the experimental groups (p < 0.001). The supplementation of broiler chickens with propolis and/or bee pollen has a strong protective effect on liver pathology in broiler chickens.

Different combinations of maternal and postnatal diet are reflected in changes of hepatic parenchyma and hepatic TNF-alpha expression in male rat offspring.

Obesity is related to increased TNF-alpha production in different tissues. TNF-alpha is connected to mitochondrial dysfunction in the liver and also development of fatty infiltration of the liver. Also, postnatal change from normal to high-fat diet causes a significant increase in TNF-alpha serum levels. The aim of this research was to determine how maternal diet and switching male offspring to a different dietary regime after lactation influences rat liver. Ten female Sprague Dawley rats at nine weeks of age were randomly divided in two groups and fed either standard laboratory chow or high-fat diet during six weeks, and then mated with the same male subject. After birth and lactation male offspring from both groups were further divided into four subgroups depending on their subsequent diet. At 22 weeks of age, the animals were weighted, sacrificed and major organs were collected and weighted. Immunohistochemistry for TNF-alpha was performed on liver, and liver samples were analyzed for pathohistological changes. The group in which mothers were fed standard chow and offspring high-fat diet had the most pronounced changes: heaviest liver, poorest histopathological findings and strongest TNF-alpha immunohistochemical staining of liver parenchyma. High-fat diet during pregnancy and lactation and switching to high-fat diet postnatally affects liver weight, histological structure and TNF-alpha expression in male offspring.

INTERLABORATORY CONCORDANCE IN HER-2 POSITIVE BREAST CANCER.

Accurate assessment of HER-2 status is essential for identifying patients who will benefit from HER-2 targeted therapy. The aim of the present study was to show results on the concordance between local and central laboratory testing results in HER-2 positive breast cancer patients. In cases with discordant findings, the immunohistochemical (IHC) and/or in situ hybridization (FISH/SISH) analysis was performed in central laboratories. A total of 104 out of 143 (72.72%) breast carcinoma cases were HER-2 positive (score 3+), while nearly 14% of tumors (20/43) showed weak (score 2+) and 12% (19/143) negative IHC staining (score 0 and 1+). After repeated IHC and ISH, 88% (126/143) were classified as HER-2 positive and 12% (17/143) as HER-2 negative cases. The results obtained are in agreement with many studies that confirmed similar discordance in HER-2 testing by IHC and/or FISH between local and central laboratory. Thus, our findings as well as those from other studies support the importance of regular quality assessment of the staining procedures performed and consistency of interpretation of HER-2 test results.

Secondary abdominal appendicular pregnancy: case report.

INTRODUCTION: The case report describes a 29-year-old nulliparous woman that was admitted at the Department of Gynecology and Obstetrics of the Clinical Hospital Osijek complaining of mild abdominal pain without vaginal discharge. CASE OUTLINE: The patient's menstrual cycle was irregular, from 30-45 days. An ultrasound examination showed suspicion of an ectopic pregnancy with a betaHCG level of 1358 IU/L. Due to the presence of liquid in the pouch of Douglas the patient underwent emergency laparoscopy, which showed the presence of tumor mass between the right Fallopian tube and the appendix. These two structures associated with adhesions corresponded to secondary implantation after spontaneous tubal abortion which was confirmed by histopathologic analysis. CONCLUSION: Laparoscopy has emerged as the"gold standard"in the diagnosis and treatment of ectopic pregnancy, in this case the secondary abdominal pregnancy. From the diagnostic point of view, all women of reproductive age should be considered pregnant until proven otherwise, also keeping in mind that ectopic pregnancies can have different locations and many clinical features.

"Signet-ring" cell invasive lobular carcinoma of the breast - accidental finding associated with intraductal papilloma: a case report.

BACKGROUND: A 57-year old woman had only unilateral milky dischardge of the right breast. Clinical and mammography findings were normal. CASE PRESENTATION: Cytological diagnosis of intraductal papilloma was established which was galactographically confitmed and patient underwent to surgery. Ductulolobular segmentectomy was made. Histopathologically beside intraductal papilloma numerous single dispread malignant "signet ring" cells in the fibrous retromammilary stroma were found. Imunohistochemically findings were: cytokeratin 8 positive, ER H-score 80, PR H-score 50, HER-2/neu negative. Diagnosis of "signet ring" cell lobular invasive carcinoma was made, followed by mastectomy, axillary limphadectomy and contra lateral breast biopsy. CONCLUSION: Residual tumor were found only in the breast tissue, while axillary lymph nodes and contra lateral breast biopsy were negative. Patient underwent to oncology therapy.

Tumor growth fraction, expression of estrogen and progesterone receptors, p53, bcl-2 and cathepsin D activity in primary ductal invasive breast carcinoma and their axillary lymph node metastases.

The aim of this paper is to determine similarities and differences between tumor cell subclones in cases of ductal invasive breast carcinoma, and which occupy primary tumor and local axillary lymph metastases. The tumor growth fraction evaluated by Ki-67 was analyzed along with the expression level of estrogen and progesterone receptors, protein p53, proto-oncogene protein bcl-2 and cathepsin D in 60 patients. Metastatic lymph node in axilla has a higher growth fraction of the tumor cells than the primary tumor (p = 0.045), as well as the higher level of bcl-2 overexpression (p = 0.014). No statistically significant difference was found in the presence of immunohistochemically identified estrogen receptors (p = 0.161) and progesterone receptors (p = 0.081) between the primary tumor and the metastatic lymph node in axilla. Likewise, no difference was found between the immunohistochemical evaluation of p53 (p = 0.356) and cathepsin D activity (p = 0.928). A higher growth fraction of the tumor cells and the higher level of bcl-2 overexpression in metastatic tumor cells indicate the more aggressive cell subclones. This study does not support the routine testing of both primary tumor and locoregional metastasis to evaluate the breast cancer hormone receptor status.

[Prognostic significance of newly formed blood vessels in the tumors of patients with breast cancer]. / Prognosticka vrijednost novostvorenih krvnih zila u tumorima bolesnica s rakom dojke.

In this study 96 specimens of ductal invasive breast carcinoma were analyzed. The following data of each patient were collected: age, tumor size, histological grade, axillary lymph node metastases, estrogen (ER) and progesterone (PR) receptor expression, percentage of cells in S-phase of cell cycle, cell ploidy status, and overall survival. From paraffin blocks 3-microns sections were stained using anti-factor VIII monoclonal antibody for detection of tumor neoangiogenesis. The number of new blood vessels/mm2 in the stained tumor tissue specimens was analyzed using a light microscope with Weibel graticule on the eyepiece. There was no statistically significant correlation of number of blood vessels/mm2 and age, axillary lymph node status, ploidy of tumor cells, size and tumor grade, ER and PR status, but there is a correlation with number of cells in the S-phase of cell cycle (p = 0.037). The cut-off value of tumor blood vessels/mm2 was 170. Univeriate analysis showed that overall survival correlated significantly with axillary lymph node involvement (p < 0.001), ER (p = 0.012) and PR (p = 0.001) status, and number of blood vessels/mm2 of tumor (p = 0.033). In multivariate analysis only axillary lymph node metastases (p = 0.015) and PR status (p = 0.026) were found to be independent and significant prognostic factors. When patients were stratified according to number of blood vessels/mm2 of tumor it was shown that those with the number of blood vessels/mm2 over 170, aged under 50 years (p = 0.011), number of cells in S-phase of cell cycle over 4% (p = 0.050), diploid tumor cells (p = 0.004), and negative PR (p = 0.059) had shorter survival than patients with tumors with less than 170 blood vessels/mm2 of tumor.