Early microvascular coronary endothelial dysfunction precedes pembrolizumab-induced cardiotoxicity. Preventive role of high dose of atorvastatin.

Immune checkpoint inhibitors (ICIs) exhibit remarkable antitumor activity and immune-related cardiotoxicity of unknown pathomechanism. The aim of the study was to investigate the ICI class-dependent cardiotoxicity in vitro and pembrolizumab's (Pem's) cardiotoxicity in vivo, seeking for translational prevention means. Cytotoxicity was investigated in primary cardiomyocytes and splenocytes, incubated with ipilimumab, Pem and avelumab. Pem's cross-reactivity was assessed by circular dichroism (CD) on biotechnologically produced human and murine PD-1 and in silico. C57BL6/J male mice received IgG4 or Pem for 2 and 5 weeks. Echocardiography, histology, and molecular analyses were performed. Coronary blood flow velocity mapping and cardiac magnetic resonance imaging were conducted at 2 weeks. Human EA.hy926 endothelial cells were incubated with Pem-conditioned media from human mononuclear cells, in presence and absence of statins and viability and molecular signaling were assessed. Atorvastatin (20 mg/kg, daily) was administered in vivo, as prophylaxis. Only Pem exerted immune-related cytotoxicity in vitro. Pem's cross-reactivity with the murine PD-1 was confirmed by CD and docking. In vivo, Pem initiated coronary endothelial and diastolic dysfunction at 2 weeks and systolic dysfunction at 5 weeks. At 2 weeks, Pem induced ICAM-1 and iNOS expression and intracardiac leukocyte infiltration. At 5 weeks, Pem exacerbated endothelial activation and triggered cardiac inflammation. Pem led to immune-related cytotoxicity in EA.hy926 cells, which was prevented by atorvastatin. Atorvastatin mitigated functional deficits, by inhibiting endothelial dysfunction in vivo. We established for the first time an in vivo model of Pem-induced cardiotoxicity. Coronary endothelial dysfunction precedes Pem-induced cardiotoxicity, whereas atorvastatin emerges as a novel prophylactic therapy.

Systems medicine dissection of chr1q-amp reveals a novel PBX1-FOXM1 axis for targeted therapy in multiple myeloma.

Understanding the biological and clinical impact of copy number aberrations (CNAs) on the development of precision therapies in cancer remains an unmet challenge. Genetic amplification of chromosome 1q (chr1q-amp) is a major CNA conferring an adverse prognosis in several types of cancer, including in the blood cancer multiple myeloma (MM). Although several genes across chromosome 1 (chr1q) portend high-risk MM disease, the underpinning molecular etiology remains elusive. Here, with reference to the 3-dimensional (3D) chromatin structure, we integrate multi-omics data sets from patients with MM with genetic variables to obtain an associated clinical risk map across chr1q and to identify 103 adverse prognosis genes in chr1q-amp MM. Prominent among these genes, the transcription factor PBX1 is ectopically expressed by genetic amplification and epigenetic activation of its own preserved 3D regulatory domain. By binding to reprogrammed superenhancers, PBX1 directly regulates critical oncogenic pathways and a FOXM1-dependent transcriptional program. Together, PBX1 and FOXM1 activate a proliferative gene signature that predicts adverse prognosis across multiple types of cancer. Notably, pharmacological disruption of the PBX1-FOXM1 axis with existing agents (thiostrepton) and a novel PBX1 small molecule inhibitor (T417) is selectively toxic against chr1q-amp myeloma and solid tumor cells. Overall, our systems medicine approach successfully identifies CNA-driven oncogenic circuitries, links them to clinical phenotypes, and proposes novel CNA-targeted therapy strategies in MM and other types of cancer.

Belantamab mafodotin, lenalidomide and dexamethasone in transplant-ineligible patients with newly diagnosed multiple myeloma: part 1 results of a phase I/II study.

Preclinical and clinical data demonstrate synergy between belantamab mafodotin (belamaf) and immunomodulatory drugs with limited overlapping toxicities. We investigated the safety and efficacy of belamaf with lenalidomide 25 mg on days 1-21 every 28 days and dexamethasone 40 mg weekly (belamaf-Rd) in transplant-ineligible patients with newly diagnosed multiple myeloma. Thirty-six patients (median age, 72.5 years) were randomized to receive belamaf at three different doses (2.5, 1.9, or 1.4 mg/kg) every 8 weeks. The dosing schedule was extended to every 12 weeks to mitigate ocular toxicity. Most common grade ≥3 adverse events were fatigue (n=21, 58.3%), rash (n=6, 16.7%), diarrhea (n=8, 22.2%) and COVID-19 (n=5, 13.9%). Grade 3-4 ocular adverse events, comprising visual acuity decline from baseline and/or keratopathy, were reported in 39/216 (18.1%), 33/244 (13.5%), and 26/207 (12.6%) ophthalmological assessments in the 2.5, 1.9, and 1.4 mg/kg cohorts, respectively. Importantly, grade 3-4 keratopathy was identified in 9/216 (4.2%), 1/244 (0.4%) and 1/207(0.5%) assessments. Most patients (32/36, 88.9%) were treated with the extended, every-12-week schedule, during which 40, 33 and 16 doses were withheld due to ocular adverse events in the 2.5, 1.9, and 1.4 mg/kg cohorts, respectively. Overall, the rates of very good partial response and better and complete response and better were 83.3% and 52.8%, respectively, without significant differences among cohorts. Over a median follow-up of 20.3 months no disease progression was reported; six patients discontinued treatment due to infection-related death (4 cases of COVID-19, 2 cases of pneumonia) and one patient withdrew consent. Based on the toxicity/efficacy balance, the recommended phase II dose was 1.9 mg/kg every 8 weeks, extended to every 12 weeks because of toxicity. In conclusion, Belamaf-Rd, with the extended schedule for belamaf, showed important clinical activity and a significant improvement of ocular adverse events with minimal impact on vision-related functioning in an elderly, non-transplant eligible population.

Low circulating tumor cell levels correlate with favorable outcomes and distinct biological features in multiple myeloma.

There is growing interest in multiple myeloma (MM) circulating tumor cells (CTCs), but their rareness in peripheral blood (PB) and inconsistency in cutoffs question their clinical utility. Herein, we applied next-generation flow cytometry in 550 bone marrow (BM) and matched PB samples to define an optimal CTC cutoff for both transplant-eligible and transplant-ineligible newly diagnosed MM (NDMM) patients. Deep phenotyping was performed to investigate unique microenvironmental features associated with CTC dissemination. CTCs were detected in 90% of patients (median 0.01%; range: 0.0002%-12.6%) and increased levels associated with adverse features. Correlations were observed between high CTC percentages and a diffused MRI pattern, a distinct BM composition characterized by altered B-cell differentiation together with an expansion of effector cells and tumor-associated macrophages, as well as a greater phenotypic dissimilarity between BM and PB clonal cells. Progression-free survival (PFS) and overall survival (OS) gradually worsened with each logarithmic increment of CTCs. Conversely, NDMM patients without CTCs showed unprecedented outcomes, with 5-year PFS and OS rates of 83% and 97%, respectively. A cutoff of 0.02% CTCs was independent of the ISS, LDH, and cytogenetics in a multivariate analysis of risk factors for PFS. The 0.02% CTC cutoff synergized with the MGUS-like phenotype and the R-ISS for improving the risk stratification systems. MRD negativity was less frequent if CTCs were ≥0.02% at diagnosis, but whenever achieved, the poor prognosis of these patients was abrogated. This study shows the clinical utility of CTC assessment in MM and provides evidence toward a consensus cutoff for risk stratification.

Central nervous system multiple myeloma: A real-world multi-institutional study of the Greek Myeloma Study Group.

Central nervous system (CNS) involvement is a rare and aggressive complication of multiple myeloma (MM). We identified 54/4352 MM patients (1.2%), who developed CNS-MM between 2000 and 2022. A matched-control group of MM patients without CNS-MM was used for comparisons. Median age was 63 years. Median time to CNS-MM was 28 months; 6/54 experienced CNS-MM at MM diagnosis. Abnormal lactate dehydrogenase (LDH), high-risk cytogenetics, and extramedullary involvement (EMI), that is, soft tissue plasmacytomas and/or plasma cell leukemia (PCL), were more frequent in CNS-MM versus controls (p < .05); 13/54 had PCL at CNS-MM. The majority had leptomeningeal infiltration (LMI) (66%); 26% had CNS-MM without systemic myeloma; EMI was the strongest predictor for CNS-MM (OR: 6.3). Median overall survival (OS) of CNS-MM patients versus controls was 43 months (95% CI: 32-54) versus 60 months (95% CI: 38-82) (p < .001); treatment of CNS-MM included mainly bortezomib/thalidomide/chemotherapy whereas 20% received novel drugs/immunotherapy combinations; 28 patients underwent cerebrospinal fluid infusions; EMI was the strongest negative predictor for post CNS-MM OS (p = .005; HR: 2.9). Treatment after 2016 predicted significantly for OS (p = .002; HR: 0.27). Median post CNS-MM OS was 4 months (95% CI: 2.6-5.4); in patients treated after 2016 median OS was 12 months. In conclusion, we have demonstrated in this large real-world series that survival of CNS-MM remains poor; however, there is a positive impact of treatment after 2016, related to the efficacy of modern anti-myeloma therapy; EMI significantly increases the probability to develop CNS-MM and the risk of post CNS-MM death, indicating a potential need for CNS prophylaxis for those patients.

Improved survival of patients with primary plasma cell leukemia with VRd or daratumumab-based quadruplets: A multicenter study by the Greek myeloma study group.

We evaluated the efficacy and prognostic impact of bortezomib-lenalidomide triplet (VRd) or daratumumab-based quadruplets (DBQ) versus previous anti-myeloma therapies, that is, bortezomib standard combinations (BSC) or conventional chemotherapy (CT), in a large cohort of patients with primary plasma cell leukemia (pPCL), including those fulfilling the revised diagnostic criteria, that is, circulating plasma cells (cPCS): ≥5%; 110 pPCL patients (M/F: 51/59; median age 65 years, range: 44-86) out of 3324 myeloma patients (3%), registered in our database between 2001 and 2021, were studied; 37% had cPCS 5%-19%; 89% received novel combinations including DBQ (21%), VRd (16%) and BSC (52%); 35% underwent autologous stem cell transplantation. 83% achieved objective responses. Treatment with VRd/DBQ strongly correlated with a higher complete response rate (41% vs. 17%; p = .008). After a median follow-up of 51 months (95% CI: 45-56), 67 patients died. Early mortality was 3.5%. Progression-free survival was 16 months (95% CI: 12-19.8), significantly longer in patients treated with VRd/DBQ versus BSC/CT (25 months, 95% CI: 13.5-36.5 vs. 13 months 95% CI: 9-16.8; p = .03). Median overall survival (OS) was 29 months (95% CI: 19.6-38.3), significantly longer in patients treated with VRd/DBQ versus BSC/CT (not reached vs. 20 months, 95% CI: 14-26; 3-year OS: 70% vs. 32%, respectively; p < .001; HzR: 3.88). In the multivariate analysis VRd/DBQ therapy, del17p(+) and PLT <100.000/µL, independently predicted OS (p < .05). Our study has demonstrated that in the real-world setting, treatment with VRd/DBQ induces deep and durable responses and is a strong prognostic factor for OS representing currently the best therapeutic option for pPCL.

Distinct type I interferon responses between younger women and older men contribute to the variability of COVID-19 outcomes: Hypothesis generating insights from COVID-19 convalescent individuals.

BACKGROUND/OBJECTIVE: Older age and male sex have been consistently found to be associated with dismal outcomes among COVID-19 infected patients. In contrast, premenopausal females present the lowest mortality among adults infected by SARS-CoV-2. The goal of the present study was to investigate whether peripheral blood type I interferon (IFN) signature and interleukin (IL)-6 serum levels -previously shown to contribute to COVID-19-related outcomes in hospitalized patients- is shaped by demographic contributors among COVID-19 convalescent individuals. PATIENTS AND METHODS: Type I IFN-inducible genes in peripheral blood, as well as serum IL-6 levels were quantified in 61 COVID-19 convalescent healthy individuals (34 females, 27 males; age range 18-70 years, mean 35.7 ± 15.9 years) who recovered from COVID-19 without requiring hospitalization within a median of 3 months prior to inclusion in the present study. Among those, 17 were older than 50 years (11 males, 6 females) and 44 equal to or less than 50 years (16 males, 28 females). Expression analysis of type I IFN-inducible genes (MX-1, IFIT-1, IFI44) was performed by real time PCR and a type I IFN score, reflecting type I IFN peripheral activity, was calculated. IL-6 and C-reactive protein levels were determined by a commercially available ELISA. RESULTS: COVID-19 convalescent individuals older than 50 years exhibited significantly decreased peripheral blood type I IFN scores along with significantly increased IL-6 serum levels compared to their younger counterparts less than 50 years old (5.4 ± 4.3 vs 16.8 ± 24.7, p = 0.02 and 10.6 ± 16.9 vs 2.9 ± 8.0 ng/L, p = 0.03, respectively). Following sex stratification, peripheral blood type I IFN score was found to be significantly higher in younger females compared to both younger and older males (22.9 ± 29.2 vs 6.3 ± 4.6 vs 4.5 ± 3.7, p = 0.01 and p = 0.002, respectively). Regarding IL-6, an opposite pattern was observed, with the highest levels being detected among older males and the lowest levels among younger females (11.6 ± 18.9 vs 2.5 ± 7.8 ng/L, p = 0.03). CONCLUSION: Constitutive higher type I IFN responses and dampened IL-6 production observed in younger women of premenopausal age, along with lower type I IFN responses and increased IL-6 levels in older males, could account for the discrete clinical outcomes seen in the two population groups, as consistently revealed in COVID-19 epidemiological studies.

The innate sensor ZBP1-IRF3 axis regulates cell proliferation in multiple myeloma.

Multiple myeloma is a malignancy of plasma cells initiated and driven by primary and secondary genetic events. However, myeloma plasma cell survival and proliferation might be sustained by non-genetic drivers. Z-DNA-binding protein 1 (ZBP1; also known as DAI) is an interferon-inducible, Z-nucleic acid sensor that triggers RIPK3-MLKL-mediated necroptosis in mice. ZBP1 also interacts with TBK1 and the transcription factor IRF3 but the function of this interaction is unclear, and the role of the ZBP1-IRF3 axis in cancer is not known. Here we show that ZBP1 is selectively expressed in late B-cell development in both human and murine cells and it is required for optimal T-cell-dependent humoral immune responses. In myeloma plasma cells, the interaction of constitutively expressed ZBP1 with TBK1 and IRF3 results in IRF3 phosphorylation. IRF3 directly binds and activates cell cycle genes, in part through co-operation with the plasma cell lineage-defining transcription factor IRF4, thereby promoting myeloma cell proliferation. This generates a novel, potentially therapeutically targetable and relatively selective myeloma cell addiction to the ZBP1-IRF3 axis. Our data also show a noncanonical function of constitutive ZBP1 in human cells and expand our knowledge of the role of cellular immune sensors in cancer biology.

Daratumumab-based therapy for patients with monoclonal gammopathy of renal significance.

Treatment of the plasma cell clone in monoclonal gammopathy of renal significance (MGRS) is necessary in order to reduce toxic immunoglobulin load to the kidneys and salvage renal function. There are limited data on the use of daratumumab in patients with MGRS. We summarize our experience with the use of daratumumab-based therapy in 25 MGRS patients, 12 of whom were previously untreated. The median follow-up of the cohort is 14 months. The best overall haematologic response in evaluable patients was complete response (CR) in five (22%), very good partial response (VGPR) in five (22%) and partial response (PR) in seven (30%) patients for an overall response rate of 74%. Two of five patients in CR and two patients with initially detectable clones, but non-measurable immunoglobulins, had undetectable minimal residual disease (MRD) with next-generation flow cytometry (NGF) after therapy. Haematologic response rate for previously untreated patients was 83% vs. 69% for previously treated and for daratumumab combinations it was 91% vs. 64%, and with CR/VGPR 82% vs. 29%, compared to daratumumab monotherapy. At six months, 12/22 (55%) patients not on dialysis achieved a reduction of proteinuria >30%, of at least 0·5 g/24 h, without an estimated glomerular filtration rate (eGFR) reduction. The toxicity was mild and predictable. In conclusion, daratumumab-based therapy is a new option for patients with MGRS.

Prognostic impact of stromal and intratumoral CD3, CD8 and FOXP3 in adjuvantly treated breast cancer: do they add information over stromal tumor-infiltrating lymphocyte density?

BACKGROUND: Tumor-infiltrating lymphocytes (TILs) and their subsets contribute to breast cancer prognosis. We investigated the prognostic impact of CD3+, CD8+ and FOXP3+ TILs in patients with early intermediate/high-risk breast cancer treated with adjuvant anthracycline-based chemotherapy within two randomized trials conducted by our Group. METHODS: We examined 1011 patients (median follow-up 130.9 months) and their tumors for total, stromal (s) and intratumoral (i) CD3, CD8 and FOXP3 lymphocyte density (counts/mm2) on tissue-microarray cores by immunohistochemistry. Morphological sTIL density on whole H&E-stained sections was also evaluated. RESULTS: The majority of TILs were CD3+. Total CD3 and CD8, sCD3 and sCD8, iCD3 and iCD8, sFOXP3 and iFOXP3 were strongly correlated (Spearman's rho values > 0.6). High individual lymphocytic subsets and sTIL density were strongly associated with high tumor grade, higher proliferation and HER2-positive and triple-negative tumors (all p values < 0.001). Higher sTIL density (10% increments), high density of almost each individual marker and all-high profiles conferred favorable prognosis. However, when adjusted for sTIL density, stromal and intratumoral lymphocytic subsets lost their prognostic significance, while higher sTIL density conferred up to 15% lower risk for relapse. Independently of sTIL density, higher total CD3+ and CD8+ TILs conferred 35% and 28% lower risk for relapse, respectively. CONCLUSIONS: Stromal and intratumoral CD3+, CD8+ and FOXP3+ TIL density do not seem to add prognostic information over the morphologically assessed sTIL density, which is worth introducing in routine histology reports.

TdT expression in normal and neoplastic sebaceous cells.

AIMS: Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase expressed in immature, normal and neoplastic, lymphoid or haematopoietic cells and in neuroendocrine carcinomas, such as Merkel cell carcinoma and small-cell carcinoma. It has not yet been described in cells of epithelial origin. After observing TdT immunoreactivity in normal sebaceous glands, we analysed its spectrum of expression in cases of sebaceous cell hyperplasia (SGH) and sebaceous cell neoplasm. METHODS AND RESULTS: Twelve cases of SGH and three cases of other benign lesions, namely sebaceoma, sebaceous adenoma, and sebaceous naevus, along with four archived cases of sebaceous cell carcinoma (SC) were collected and stained with TdT antibody. In addition, tissue microarrays were constructed from 11 cases of basal cell carcinoma (BCC) and 10 cases of squamous cell carcinoma (SCC), which had nine evaluable cases each, and, after carcinoma type confirmation with immunostaining for epithelial membrane antigen, TdT immunohistochemistry was performed. All cases of SGH and sebaceous cell neoplasm were positive for TdT. The staining intensity was variable, being often weak to moderate in a significant proportion of cells, apart from one case of SC and the case of sebaceous naevus, which were only focally positive. No BCCs and only one SCC showed immunoreactivity. CONCLUSIONS: TdT protein can be found in cells of epithelial origin and specifically sebaceous cells, both benign and malignant. It can be hypothesized that this expression is due to sebaceous cell differentiation as a prelude to apoptosis and holocrine secretion. Additional studies are needed to further elucidate its biological role.

MYC copy gain, chromosomal instability and PI3K activation as potential markers of unfavourable outcome in trastuzumab-treated patients with metastatic breast cancer.

BACKGROUND: There is an unmet need for more efficient patient stratification for receiving trastuzumab in the metastatic breast cancer (mBC) setting, since only part of such patients benefit from the addition of this agent to chemotherapy. The aim of this study was to investigate the prognostic value of biomarkers including MYC and MET in mBC patients treated with trastuzumab-based regimens. METHODS: mBC patients, locally tested as HER2-positive, treated with trastuzumab and chemotherapy between 1998 and 2010 were evaluated. Paraffin tumors (n = 229) were retrospectively centrally assessed by immunohistochemistry (IHC) for HER2, ER, PgR and Ki67; fluorescence in situ hybridization (FISH) for HER2, TOP2A and centromere (CEN) 17, MYC and CEN8, MET and CEN7; qPCR for MYC, MET copy number (CN); and, for PI3K activation (PIK3CA mutations; PTEN and phospho-mTOR protein expression). Increased CEN CN was assessed based on normal cut-offs. Time to progression (TTP) and survival were evaluated from the initiation of trastuzumab as first line treatment. RESULTS: Among all tumors, 90 were HER2-negative upon central testing (ambiguous HER2) and the rest were true HER2-positive. Further, 156 patients presented with mBC upon relapse of pre-treated disease (R-mBC) and 65 were diagnosed at stage IV (de novo mBC). Concordance between FISH and qPCR on gene CN status was fair for MYC (Kappa = 0.458) and absent for MET. The presence of MYC CN gain with qPCR and the absence of PI3K activation were infrequent events (7 and 8 % of evaluable tumors, respectively), while 41 % of tumors had increased CEN CN in one or more chromosomes, indicative of chromosomal instability. The most consistent finding in the entire cohort and in the above patient subgroups with respect to outcome was the unfavourable effect of MYC CN gain, which was retained upon multivariable analysis (e.g., survival in the entire cohort, HR 6.02; 95 % CI 2.67-13.6; p < 0.001). Further unfavourable prognosticators were increased CEN CN in one chromosome in R-mBC but not in de novo mBC (multivariable interaction p = 0.048), PI3K activation in R-mBC (multivariable p = 0.004) and increased Ki67 for patient TTP. CONCLUSIONS: MYC gene copies, centromere status and PI3K activation may adversely impact trastuzumab treated mBC patient outcome and seem worthy validating in larger series.

Bilateral Aleukemic Myeloid Sarcoma of the Eyelids With Indolent Course.

Leukemic infiltrates may be seen in the skin in the absence of detectable bone marrow involvement. Leukemia cutis may exceptionally occupy the eyelids. An unusual case of a 58-year-old man presenting bilateral erythematous eyelid lesions, proven to be aleukemic leukemia cutis, is reported. Biopsy was conducted and hematoxylin/eosin stained sections were histologically evaluated. Immunohistochemistry was also performed.Light microscopy revealed cutaneous infiltration by a neoplastic population consisting of medium-sized cells. These cells infiltrated the overlying epidermis leading to focal microulcerations. The morphological and immunohistochemical characteristics of the neoplastic population were compatible with myeloid leukemia cutis. The bone marrow biopsy was normocellular for the patient's age. Although chemotherapy was advised, the patient refused any treatment. He remains free of leukemia or evolution of eyelid lesions approximately 1 year after diagnosis. Leukemia cutis of the eyelids is a rare manifestation of acute leukemia and may remain aleukemic in adults for an indefinite period of time.

Immunophenotypic analysis reveals heterogeneity and common biologic aspects in monoclonal B-cell lymphocytosis.

Monoclonal B-cell lymphocytosis (MBL) is the presence of small B-cell clones in the peripheral blood of healthy subjects. Most MBL have the characteristic phenotype of chronic lymphocyte leukemia (chronic lymphocytic leukemia (CLL)-like MBL), and depending on the number of monoclonal B-cells, may characterize a preclinical stage of the CLL. However, there are also MBL with an atypical (CD5(+) CD20(+/bright) CD23(dim/-) ) or a CD5(neg) phenotype, which remain largely unexplored. We performed an extended immunophenotypic, cytogenetic, and hematologic analysis in 75 CLL-like, 39 atypical, 50 CD5(neg) , and 7 biphenotypic MBL cases to detect differences or similarities among the MBL subsets. The phenotypic analysis showed expression variations in many surface markers and a wide spectrum of disease-specific phenotypes within each MBL subtype. Interphase fluorescent in situ hybridization analysis showed a different panel of aberrations according to the phenotype. Overall, del(13q14) and +12 were the most common abnormalities (39%), whereas del(11q13), del(17p13), and del(6q23) were detected only in 3, 1, and 0 cases, respectively. A comparison of MBL with overt chronic lymphoproliferations revealed common aspects in the preclinical state, regarding both the kind of cytogenetic aberrations detected and the lymphocyte composition. Our findings highlight not only the heterogeneity among MBL subsets but also indicate common biologic features which differentiate MBL from clinical disease.

alphaB-crystallin is a marker of aggressive breast cancer behavior but does not independently predict for patient outcome: a combined analysis of two randomized studies.

BACKGROUND: alphaB-crystallin is a small heat shock protein that has recently been characterized as an oncoprotein correlating with the basal core phenotype and with negative prognostic factors in breast carcinomas. The purpose of this study was to evaluate alphaB-crystallin with respect to clinicopathological parameters and the outcome of patients with operable high-risk breast cancer. METHODS: A total of 940 tumors were examined, derived from an equal number of patients who had participated in two randomized clinical trials (paclitaxel-containing regimen in 793 cases). Immunohistochemistry for ER, PgR, HER2, Ki67, CK5, CK14, CK17, EGFR, alphaB-crystallin, BRCA1 and p53 was performed. BRCA1 mutation data were available in 89 cases. RESULTS: alphaß-crystallin was expressed in 170 cases (18.1%) and more frequently in triple-negative breast carcinomas (TNBC) (45% vs. 14.5% non-TNBC, p < 0.001). alphaB-crystallin protein expression was significantly associated with high Ki67 (Pearson chi-square test, p < 0.001), p53 (p = 0.002) and basal cytokeratin protein expression (p < 0.001), BRCA1 mutations (p = 0.045) and negative ER (p < 0.001) and PgR (p < 0.001). Its overexpression, defined as >30% positive neoplastic cells, was associated with adverse overall survival (Wald's p = 0.046). However, alphaB-crystallin was not an independent prognostic factor upon multivariate analysis. No interaction between taxane-based therapy and aß-crystallin expression was observed. CONCLUSIONS: In operable high-risk breast cancer, alphaB-crystallin protein expression is associated with poor prognostic features indicating aggressive tumor behavior, but it does not seem to have an independent impact on patient survival or to interfere with taxane-based therapy. TRIAL REGISTRATIONS: ACTRN12611000506998 (HE10/97 trial) and ACTRN12609001036202 (HE10/00 trial).

Molecular detection of Helicobacter pylori in a large Mediterranean river, by direct viable count fluorescent in situ hybridization (DVC-FISH).

Although the precise route and mode of transmission of Helicobacter pylori are still unclear, molecular methods have been applied for the detection of H. pylori in environmental samples. In this study, we used the direct viable count fluorescent in situ hybridization (DVC-FISH) method to detect viable cells of H. pylori in the River Aliakmon, Greece. This is the longest river in Greece, and provides potable water in metropolitan areas. H. pylori showed positive detection for 23 out of 48 water samples (47.9%), while no seasonal variation was found and no correlation was observed between the presence of H. pylori and indicators of fecal contamination. Our findings strengthen the evidence that H. pylori is waterborne while its presence adds to the potential health hazards of the River Aliakmon.

Prebiotics increase iron absorption and reduce the adverse effects of iron on the gut microbiome and inflammation: a randomized controlled trial using iron stable isotopes in Kenyan infants.

BACKGROUND: Iron fortificants tend to be poorly absorbed and may adversely affect the gut, especially in African children. OBJECTIVE: We assessed the effects of prebiotic galacto-oligosaccharides/fructo-oligosaccharides (GOS/FOS) on iron absorption and gut health when added to iron-fortified infant cereal. METHODS: We randomly assigned Kenyan infants (n = 191) to receive daily for 3 wk a cereal containing iron and 7.5 g GOS/FOS (7.5 g+iron group), 3 g (3-g+iron group) GOS/FOS, or no prebiotics (iron group). A subset of infants in the 2 prebiotic+iron groups (n = 66) consumed 4 stable iron isotope-labeled test meals without and with prebiotics, both before and after the intervention. Primary outcome was fractional iron absorption (FIA) from the cereal with or without prebiotics regardless of dose, before and after 3 wk of consumption. Secondary outcomes included fecal gut microbiota, iron and inflammation status, and effects of prebiotic dose. RESULTS: Median (25th-75th percentiles) FIAs from meals before intervention were as follows: 16.3% (8.0%-27.6%) without prebiotics compared with 20.5% (10.4%-33.4%) with prebiotics (Cohen d = 0.53; P < 0.001). FIA from the meal consumed without prebiotics after intervention was 22.9% (8.5%-32.4%), 41% higher than from the meal without prebiotics before intervention (Cohen d = 0.36; P = 0.002). FIA from the meal consumed with prebiotics after intervention was 26.0% (12.2%-36.1%), 60% higher than from the meal without prebiotics before intervention (Cohen d = 0.45; P = 0.007). After 3 wk, compared with the iron group, the following results were observed: 1) Lactobacillus sp. abundances were higher in both prebiotic+iron groups (P < 0.05); 2) Enterobacteriaceae sp. abundances (P = 0.022) and the sum of pathogens (P < 0.001) were lower in the 7.5-g+iron group; 3) the abundance of bacterial toxin-encoding genes was lower in the 3-g+iron group (false discovery rate < 0.05); 4) fecal pH (P < 0.001) and calprotectin (P = 0.033) were lower in the 7.5-g+iron group. CONCLUSIONS: Adding prebiotics to iron-fortified infant cereal increases iron absorption and reduces the adverse effects of iron on the gut microbiome and inflammation in Kenyan infants. This trial was registered at clinicaltrials.gov as NCT03894358.

Evaluation of the prognostic role of centromere 17 gain and HER2/topoisomerase II alpha gene status and protein expression in patients with breast cancer treated with anthracycline-containing adjuvant chemotherapy: pooled analysis of two Hellenic Cooperative Oncology Group (HeCOG) phase III trials.

BACKGROUND: The HER2 gene has been established as a valid biological marker for the treatment of breast cancer patients with trastuzumab and probably other agents, such as paclitaxel and anthracyclines. The TOP2A gene has been associated with response to anthracyclines. Limited information exists on the relationship of HER2/TOP2A gene status in the presence of centromere 17 (CEP17) gain with outcome of patients treated with anthracycline-containing adjuvant chemotherapy. METHODS: Formalin-fixed paraffin-embedded tumor tissue samples from 1031 patients with high-risk operable breast cancer, enrolled in two consecutive phase III trials, were assessed in a central laboratory by fluorescence in situ hybridization for HER2/TOP2A gene amplification and CEP17 gain (CEP17 probe). Amplification of HER2 and TOP2A were defined as a gene/CEP17 ratio of >2.2 and ≥2.0, respectively, or gene copy number higher than 6. Additionally, HER2, TopoIIa, ER/PgR and Ki67 protein expression was assessed by immunohistochemistry (IHC) and patients were classified according to their IHC phenotype. Treatment consisted of epirubicin-based adjuvant chemotherapy followed by hormonal therapy and radiation, as indicated. RESULTS: HER2 amplification was found in 23.7% of the patients and TOP2A amplification in 10.1%. In total, 41.8% of HER2-amplified tumors demonstrated TOP2A co-amplification. The median (range) of HER2, TOP2A and CEP17 gain was 2.55 (0.70-45.15), 2.20 (0.70-26.15) and 2.00 (0.70-26.55), respectively. Forty percent of the tumors had CEP17 gain (51% of those with HER2 amplification). Adjusting for treatment groups in the Cox model, HER2 amplification, TOP2A amplification, CEP17 gain and HER2/TOP2A co-amplification were not associated with time to relapse or time to death. CONCLUSION: HER2 amplification, TOP2A amplification, CEP17 gain and HER2/TOP2A co-amplification were not associated with outcome in high-risk breast cancer patients treated with anthracycline-based adjuvant chemotherapy. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (ANZCTR) ACTRN12611000506998 and ACTRN12609001036202.

Non-Traditional Cardiovascular Risk Factors in Adolescents with Obesity and Metabolic Syndrome May Predict Future Cardiovascular Disease.

Obesity in adolescence is associated with significant morbidity and predisposes adolescents to the development of cardiovascular disease (CVD). Although a number of traditional CVD risk factors have been identified in youth, limited data exist regarding non-traditional CVD risk factors. In 89 adolescents with metabolic syndrome (MetS), with 60 age-, gender-, and BMI-matched controls, we determined the non-traditional CVD risk factors (hs-CRP, TG/HDL ratio, ApoB/ApoA1 ratio, NAFLD) in order to investigate whether they may be used as biomarkers for predicting future CVD, and we evaluated their response to the implementation of a multidisciplinary, personalized, lifestyle intervention program for 1 year. We demonstrated that the TG/HDL ratio, IL-2, IL-6, IL-17A, and INF-γ were significantly increased in subjects with MetS than in controls, and may be used as biomarkers to predict future CVD. Subjects with MetS had an increased mean carotid intima-media thickness (cIMT) and prevalence of NAFLD than the controls, while the prevalence of NAFLD correlated strongly with cIMT and IL-6 concentrations. Most of the non-traditional cardiovascular risk factors improved following the implementation of a lifestyle intervention program. These findings indicate that adolescents with MetS may have a greater risk for developing atherosclerosis early in life, while early lifestyle intervention is crucial for preventing the arteriosclerotic process in youth.