Altered Expression of RB and pRB in Tissue Arrays of Primary Breast Cancers and Matched Axillary Lymph Node Metastases.

Objectives: The retinoblastoma (RB) pathway is crucial in the development and progression of many cancers. To better understand the biology of progressive breast cancer (BC), we examined protein expression of the RB pathway in primary BCs and matched axillary lymph node metastases (LM). Methods: Immunohistochemistry was used to evaluate cyclin D1, CDK4/6, RB, phosphorylated RB (pRB), and E2F1 expression in tissue arrays containing cores of 50 primary BCs and matched LM. The number of positive tumor cells and staining intensity were scored. Results: The proteins were localized in the nucleus, while CDK6 was detected in the cytoplasm and CDK4 was found in both. pRB and E2F1 showed higher expression in matched LM than in primary tumors. Expression of these proteins differed significantly by the percentage of positive tumor cells, while proteins in the proximal portion of the RB pathway showed no significant differences. The main path of alteration consisted of high pRB in primary BC, remaining pRB high in the majority of LM, variations occurring in fewer cases. All matched LM of the few primary tumors that had unaltered RB and pRB expression showed changes in RB or pRB expression. Conclusion: Expression of pRB and E2F1 was significantly higher in LM than in primary BC. A majority of cancers with LM showed altered RB or pRB expression, suggesting that proteins downstream in the RB pathway play a critical role in metastatic BC and disease progression. So looking at the RB pathway could be an option for chemotherapy decisions in patients with only few LM.

Heterosubtypic, cross-reactive immunity to human Cytomegalovirus glycoprotein B.

Cytomegalovirus (CMV) genome is highly variable and heterosubtypic immunity should be considered in vaccine development since it can enhance protection in a cross-reactive manner. Here, we developed a protein array to evaluate heterosubtypic immunity to CMV glycoprotein B (gB) in natural infection and vaccination. DNA sequences of four antigenic domains (AD1, AD2, AD4/5, and AD5) of gB were amplified from six reference and 12 clinical CMV strains, and the most divergent genotypes were determined by phylogenetic analysis. Assigned genotypes were in vitro translated and immobilized on protein array. Then, we tested immune response of variable serum groups (primarily infected patients, reactivated CMV infections and healthy individuals with latent CMV infection, as well gB-vaccinated rabbits) with protein in situ array (PISA). Serum antibodies of all patient cohorts and gB-vaccinated rabbits recognized many genetic variants of ADs on protein array, including but not limited to the subtype of infecting strain. High-grade cross-reactivity was observed. In several patients, we observed none or neglectable immune response to AD1 and AD2, while the same patients showed high antibody response to AD4/5 and AD5. Among the primary infected patients, AD5 was the predominant AD, in antibody response. The most successful CMV vaccine to date contains gB and demonstrates only 50% efficacy. In this study, we showed that heterosubtypic and cross-reactive immunity to CMV gB is extensive. Therefore, the failure of CMV gB vaccines cannot be explained by a highly, strain-specific immunity. Our observations suggest that other CMV antigens should be addressed in vaccine design.

Oral Abundance of Actinomyces spp. in Breast Cancer Patients.

OBJECTIVES: Pathophysiology of medication-related osteonecrosis of the jaw (MRONJ) is still unclear, and disease development is associated with adverse reaction of bisphosphonates and denosumab, and Actinomyces spp. as well. In this study, we evaluated the abundance of Actinomyces spp. in breast cancer patients undergoing chemotherapy compared to healthy controls. METHODS: Oropharyngeal samples were collected from treatment-naive early-stage breast cancer patients, who were scheduled for standard of care therapy (eight samples throughout chemotherapy, one prior to radiotherapy and one after a year of start), as well as from healthy controls at matched timepoints. We quantified Actinomyces spp. in the samples with a highly sensitive and specific quantitative polymerase chain reaction. RESULTS: Twenty-one patients and 16 healthy subjects were enrolled. Forty-eight percent of patients suffered from estrogen receptor-positive/progesterone receptor-positive or -negative/human epidermal growth factor receptor 2 (HER2)-negative disease, 38% were HER2-positive, and 14% were triple-negative. Comparison of Actinomyces spp. loads in cancer patients and healthy controls did not reveal significant difference. Fluctuations on bacterial quantity were observed in both groups over time. Tumor receptor status or different chemotherapy schemes of patients were not correlated with a particular pattern on abundance of Actinomyces spp. CONCLUSIONS: We suggest that Actinomyces spp. are not the initiative factors in MRONJ development. These bacteria are not altered in abundance during chemotherapy, but they behave opportunistic when there is a bone disruption in the oropharynx in the first place caused by antiresorptive drugs or dental trauma and proliferate in their new niche. Thus, Actinomyces spp. plays a latter role in MRONJ development, rather than a primary causative one.

Cytomegalovirus Infection Downregulates Vitamin D Receptor in Patients Undergoing Hematopoietic Stem Cell Transplantation.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative option for patients with hematologic diseases but is associated with high mortality and morbidity. Cytomegalovirus (CMV) infection is common in HSCT patients and modulates vitamin D metabolism in vitro. We aimed at validating CMV-associated vitamin D metabolism in vivo in HSCT. METHODS: Patients treated for significant CMV viremia after HSCT were evaluated for CMV load before, during, and after antiviral treatment. RNA was isolated from whole-blood samples to test for regulation of key components of the vitamin D receptor (VDR) pathway during different phases of CMV viremia. RESULTS: CMV viremia developed a mean time of 102 (±34) d post-HSCT. Maximum levels of CMV-DNA reached a mean of 5668 (±7257) copies/mL. VDR expression was downregulated to a mean of 64.3% (±42.5%) relative to the VDR expression pre-CMV viremia (P = 0.035) and lagged in recovery following antiviral treatment. Toll-like receptor (TLR) 2 mRNA was upregulated to 225.4% during CMV viremia relative to the expression pre-CMV viremia (P = 0.012) but not TLR6/7/8 and the TLR-adaptor protein MyD88. Levels of 25-OH vitamin D were reduced in all viremic patients (48.0 ± 4.8 versus 25.1 ± 3.7 ng/mL) and were even lower after periods of CMV viremia compared with the control group (48.3 ± 3.5 versus 17.8 ± 1.8 ng/mL; P = 0.008). CONCLUSIONS: CMV viremia is associated with significant dysregulation of vitamin D metabolism in HSCT patients.

Association of Cytokeratin 5 and Claudin 3 expression with BRCA1 and BRCA2 germline mutations in women with early breast cancer.

BACKGROUND: It is important to identify biomarkers associated with BRCA mutation in women with early breast cancer (BC) to improve early identification of mutation carriers. Thus, in this study, we examined the protein expression of claudin (CLDN) 3, CLDN4, CLDN7, and E-cadherin. Moreover, we analyzed additional histopathological variables and their associations in familial BC. METHODS: Immunohistochemical analysis for CLDNs and E-cadherin was performed on 237 BC cases of three different subsets of BC tumors: 62 from BRCA1 mutation carriers, 59 from BRCA2 mutation carriers, and 116 tumors from patients with BRCA wild type (WT) as controls. Histopathological data were also analyzed in the different subgroups. Logistic regression and receiver operation characteristic (ROC) curve were conducted to investigate factors associated with BRCA tumors. RESULTS: Expression of CLDN3 positively correlated with BRCA-mutated BC. CLDN3 was expressed in 58% of BRCA1-mutated tumors compared to only 7% in BRCA2-mutated tumors (p < 0.001) and 1% in WT tumors (p < 0.001). CK5 and CK14 expression were also more likely to arise in BRCA1 tumors (44 and 16%, respectively) than in the control group (8 and 4%) (p < 0.001, p = 0.012, respectively). We also found a significantly higher proportion of CK5+ among BRCA1 tumors (44%) in comparison with BRCA2-related BC (8%) (p < 0.001). In addition, there was a significant difference between both groups regarding CK14: positive expression in 16 and 5%, respectively (p = 0.030). CK5 and CK14 did not differ between the BRCA2 group and the WT tumors significantly. In a multivariate regression model, expression of CK5 (Odds ratio (OR): 6.46; 95% confidence interval (CI): 1.52-27.43; p = 0.011), and CLDN3 (OR: 200.48; 95% CI: 21.52-1867.61; p < 0.001) were associated with BRCA1 mutation status. CONCLUSIONS: Our data suggests that CLDN3, CK5, and CK14 in combination with ER, PR and HER2 are associated with BRCA1 mutation status.

Differential Claudin 3 and EGFR Expression Predicts BRCA1 Mutation in Triple-Negative Breast Cancer.

BRCA-1 mutation-associated triple-negative breast cancer (TNBC) has been hypothesized to exhibit a phenotype that is distinct from non-mutation carriers. We have analyzed immunohistochemically detected cytokeratins 5 and 14, epidermal growth factor receptor (EGFR), claudin (CLDN) 3, 4, and 7, and E-cadherin in 57 TNBC (32 BRCA1 and 8 BRCA2 tumors, 17 WT tumors). Positive staining of CLDN3 and negative EGFR expression in TNBC are associated with a BRCA1 mutation. EGFR and CLDN3 expression was able to predict the presence of BRCA1 mutation (area under curve 0.802, p < 0.001). This could help in guiding the decision for BRCA testing.

The Human Gastric Microbiome Is Predicated upon Infection with Helicobacter pylori.

The human gastric lumen is one of the most hostile environments of the human body suspected to be sterile until the discovery of Helicobacter pylori (H.p.). State of the art next generation sequencing technologies multiply the knowledge on H.p. functional genomics as well as on the colonization of supposed sterile human environments like the gastric habitat. Here we studied in a prospective, multicenter, clinical trial the 16S rRNA gene amplicon based bacterial microbiome in a total of 30 homogenized and frozen gastric biopsy samples from eight geographic locations. The evaluation of the samples for H.p. infection status was done by histopathology and a specific PCR assay. CagA status was determined by a CagA-specific PCR assay. Patients were grouped accordingly as H.p.-negative, H.p.-positive but CagA-negative and H.p.-positive and CagA-positive (n = 10, respectively). Here we show that H.p. infection of the gastric habitat dominates the gastric microbiota in most patients and is associated with a significant decrease of the microbial alpha diversity from H.p. negative to H.p. positive with CagA as a considerable factor. The genera Actinomyces, Granulicatella, Veillonella, Fusobacterium, Neisseria, Helicobacter, Streptococcus, and Prevotella are significantly different between the H.p.-positive and H.p.-negative sample groups. Differences in microbiota found between CagA-positive and CagA-negative patients were not statistically significant and need to be re-evaluated in larger sample cohorts. In conclusion, H.p. infection dominates the gastric microbiome in a multicentre cohort of patients with varying diagnoses.

Human cytomegalovirus infection downregulates vitamin-D receptor in mammalian cells.

Vitamin D (VD) is essential for the human body and involved in a wide variety of critical physiological processes including bone, muscle, and cardiovascular health, as well as innate immunity and antimicrobial responses. Here, we elucidated the significance of the VD system in cytomegalovirus (CMV) infection, which is one of the most common opportunistic infections in immunocompromised or -suppressed patients. We found that expression of vitamin D receptor (VDR) was downregulated in CMV-infected cells within 12h [hrs] post infection [p.i.] to 12% relative to VDR expression in mock-infected fibroblasts and did not recover during the CMV replication cycle of 96h. None of the biologically active metabolites of VD, cholecalciferol, calcidiol, or calcitriol, inhibit CMV replication significantly in human fibroblasts. In a feedback loop, expression of CYP24A1 dropped to 3% by 12h p.i. and expression of CYP27B1 increased gradually during the replication cycle of CMV to 970% probably as a consequence of VDR inhibition. VDR expression was not downregulated during influenza virus or adenovirus replication. The potent synthetic vitamin D analog EB-1089 was not able to inhibit CMV replication or antagonize its effect on VDR expression. Only CMV replication, and none of the other viral pathogens evaluated, inhibited the vitamin D system in vitro. In view of the pleiotropism of VDR, CMV-mediated downregulation may have far-reaching virological, immunological, and clinical implications and thus warrant further evaluations in vitro and in vivo.

Chronic lymphocytic leukemia patients have a preserved cytomegalovirus-specific antibody response despite progressive hypogammaglobulinemia.

Chronic lymphocytic leukemia (CLL) is characterized by progressive hypogammaglobulinemia predisposing affected patients to a variety of infectious diseases but paradoxically not to cytomegalovirus (CMV) disease. Moreover, we found reactivity of a panel of CLL recombinant antibodies (CLL-rAbs) encoded by a germ-line allele with a single CMV protein, pUL32, despite differing antibody binding motifs. To put these findings into perspective, we studied prospectively relative frequency of viremia, kinetics of total and virus-specific IgG over time, and UL32 genetic variation in a cohort of therapy-naive patients (n=200). CMV-DNA was detected in 3% (6/200) of patients. The decay of total IgG was uniform (mean, 0.03; SD, 0.03) and correlated with that of IgG subclasses 1-4 in the paired samples available (n=64; p<0.001). Total CMV-specific IgG kinetics were more variable (mean, 0,02; SD, 0,06) and mean decay values differed significantly from those of total IgG (p=0.034). Boosts of CMV-specific antibody levels were observed in 49% (22/45) of CMV-seropositive patients. In contrast, VZV- and EBV-specific IgG levels decayed in parallel with total IgG levels (p=0.003 and p=0.001, respectively). VZV-specific IgG even became undetectable in 18% (9/50) of patients whereas CMV-specific ones remained detectable in all seropositive patients. The observed CMV-specific IgG kinetics were predicated upon the highly divergent kinetics of IgG specific for individual antigens - glycoprotein B-specific IgG were boosted in 51% and pUL32-specific IgG in 32% of patients. In conclusion, CLL patients have a preserved CMV-specific antibody response despite progressive decay of total IgG and IgG subclasses. CMV-specific IgG levels are frequently boosted in contrast to that of other herpesviruses indicative of a higher rate of CMV reactivation and antigen-presentation. In contrast to the reactivity of multiple different CLL-rAbs with pUL32, boosts of humoral immunity are triggered apparently by other CMV antigens than pUL32, like glycoprotein B.