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Background Patients hospitalised for COVID-19 may present with or acquire bacterial or fungal infections that can affect the course of the disease. The aim of this study was to describe the microbiological characteristics of laboratory-confirmed infections in hospitalised patients with severe COVID-19. Methods We reviewed the hospital charts of a sample of patients deceased with COVID-19 from the Italian National COVID-19 Surveillance, who had laboratory-confirmed bacterial or fungal bloodstream infections (BSI) or lower respiratory tract infections (LRTI), evaluating the pathogens responsible for the infections and their antimicrobial susceptibility. Results Among 157 patients with infections hospitalised from February 2020 to April 2021, 28 (17.8%) had co-infections (≤ 48 h from admission) and 138 (87.9%) had secondary infections (> 48 h). Most infections were bacterial; LRTI were more frequent than BSI. The most common co-infection was pneumococcal LRTI. In secondary infections, Enterococci were the most frequently recovered pathogens in BSI (21.7% of patients), followed by Enterobacterales, mainly K. pneumoniae, while LRTI were mostly associated with Gram-negative bacteria, firstly Enterobacterales (27.4% of patients, K. pneumoniae 15.3%), followed by A. baumannii (19.1%). Fungal infections, both BSI and LRTI, were mostly due to C. albicans. Antibiotic resistance rates were extremely high in Gram-negative bacteria, with almost all A. baumannii isolates resistant to carbapenems (95.5%), and K. pneumoniae and P. aeruginosa showing carbapenem resistance rates of 59.5% and 34.6%, respectively. Conclusions In hospitalised patients with severe COVID-19, secondary infections are considerably more common than co-infections, and are mostly due to Gram-negative bacterial pathogens showing a very high rate of antibiotic resistance.

Aims and background Patients with metastatic breast cancer previously treated with anthracyclines for advanced disease are usually refractory to any further treatment with anthracyclines and have a poor prognosis. Therefore, new drugs or new combinations of drugs are needed. One approach has been to focus on the type of chemotherapy with low toxicity that preserves quality of life during treatment, such as weekly drug administration. Study design We designed a dose-finding study to determine the maximum tolerated dose of gemcitabine plus docetaxel, given on a weekly schedule in metastatic breast cancer previously treated with anthracyclines. Three escalating doses of gemcitabine (900, 1000 and 1100 mg/m2) on days 1 and 8 in combination with a fixed dose of docetaxel, 35 mg/m2 on days 1 and 8 were planned. Dose-limiting toxicity included grade >3 hematologic toxicity, grade >2 stomatitis, asthenia, diarrhea or organ-specific toxicity (except alopecia). Dose escalation was stopped if 1 out of 3 patients at any dose level experienced dose-limiting toxicity. Results Nine patients received a mean of 5.1 (range, 1–9) cycles. Gastrointestinal and leukopenia were the main dose-limiting toxicity. No patient experienced dose-limiting toxicity at dose level 1; at dose level 2, 2 out of 3 patients had dose-limiting toxicity and 3 additional patients treated at dose level 2 confirmed that the maximum tolerated dose had been reached. Conclusions The recommended gemcitabine dose in combination with docetaxel (35 mg/m2 for a phase II study) was established at 900 mg/m2.

Purpose: Activation of the MDR1 upstream promoter (USP) has been described previously in four lymphoblastic leukemia patients, where it is the major MDR1 promoter associated with P-glycoprotein overexpression. We asked whether MDR1 USP-derived transcripts were also present in breast carcinoma and assessed their potential as a biomarker. Experimental Design: We developed a sensitive method for detecting transcripts derived from the MDR1 USP and used it to identify MDR1 USP-derived transcripts in cell model systems, in 61 breast carcinoma biopsies of the primary tumor, and in isolated malignant epithelial cells both from the primary tumor and from the associated invaded lymph nodes. Results: The MDR1 USP was not active in several independent leukemic and breast cancer cell lines or nucleated peripheral blood cells (n = 9). However, transcripts derived from the MDR1 USP were detected in some drug-resistant cell lines and a high proportion of primary breast tumors (71.6%; n = 61), whereas they were present at low frequency in normal breast tissue (10%; n = 10). Activation of MDR1 USP was not due to chromosomal amplifications or rearrangements at the MDR1 locus. Transcription from the MDR1 USP correlated with metastatic node invasion [N = 0–3 versus N > 3 (N = number of lymph nodes invaded); Fisher’s exact test, P = 0.011] and was detected in malignant epithelial cells from the primary tumor and those that metastasized to the lymph nodes. Conclusions: MDR1 USP activation is a surrogate marker for breast carcinoma progression and can be used as a marker to study breast cancer susceptibility.

Two novel estrogen receptor beta (ERbeta) mRNA isoforms that diverge in their 5'-untranslated regions, ERbeta mRNA (0K-1) and ERbeta mRNA (0N-1), have recently been identified. This indicates that transcription of the human ERbeta gene occurs from at least two different promoters, named promoter 0K and promoter 0N. The aim of this study was to investigate the expression of ERbeta isoforms in primary cultures of normal breast epithelial cells, a panel of breast cancer cell lines and in normal breast and breast cancer tissues; and to examine whether methylation of the two ERbeta promoters is involved in regulation of ERbeta gene expression. Using quantitative real-time PCR techniques, we found that ERbeta mRNA levels were significantly lower in breast cancer cell lines than in primary cultures of normal breast epithelial cells. Bisulfite genomic sequencing analysis revealed that two promoters of the ERbeta gene exhibit distinct methylation patterns. Promoter 0N was unmethylated in normal breast epithelial cells, but extensively methylated in breast cancer cell lines. In contrast, promoter 0K was unmethylated in both normal and malignant breast epithelial cells. We demonstrated a significant correlation between promoter 0N hypermethylation and loss of ERbeta mRNA expression in breast cancer cell lines. Treatment of breast cancer cells with demethylating agent effectively reactivated the expression of ERbeta mRNA. The observations from the cell lines were also reflected in primary breast cancer tumors. Thus, expression of ERbeta mRNA in breast tumors was found to be inversely associated with the degree of methylation of promoter 0N. Our results suggest that a decreased level of ERbeta mRNA may be associated with breast tumorigenesis, and that DNA methylation is an important mechanism for ERbeta gene silencing in breast cancer.

Pegylated liposomal doxorubicin (PLD) has the advantage of delivering active anthracycline directly to the tumor site, while exposing the patient to a lesser degree of doxorubicin-associated toxicities. Recently, a regimen in which paclitaxel is infused weekly over 1 h produced substantial antitumor activity with little myelosuppression. We designed a phase II trial to study the efficacy and toxicity of 10 mg/m(2) PLD on Days 1, 8 and 15, plus 70 mg/m(2) paclitaxel weekly in patients with untreated metastatic breast cancer and a high risk of cardiotoxicity. The study included 35 patients, with 31 (88.5%) evaluable for efficacy and 35 (100%) for toxicity. A total of 28 patients (80%) had two or more sites of disease. Overall, 4 complete and 16 partial responses were noted with an overall response rate of 64.5%, with 6 cases of stable and 5 cases of progressive disease. Toxicity was found to be manageable in that the only grade 3-4 side effects recorded were palmar-plantar erythrodysesthesia, 8.5%; mucositis, 2.8%; leucopenia, 12.5%; anemia, 2.8% and AST/ALT, 2.8%. No cardiotoxicity was observed. In conclusion, weekly PLD plus paclitaxel appears to be a well-tolerated and effective approach for metastatic breast cancer patients with a high risk of cardiotoxicity.

Purpose: Oncolytic herpes simplex virus type 1 (HSV-1) vectors show considerable promise as agents for cancer therapy. We have developed a novel recombinant HSV-1 virus (JS1/34.5−/47−) for purging of occult breast cancer cells from bone marrow of patients. Here, we evaluate the therapeutic efficacy of this oncolytic virus. Experimental Design: Electron microscopy was used to determine whether human breast cancer and bone marrow cells are permissive for JS1/34.5−/47− infection. Subsequently, the biological effects of JS1/34.5−/47− infection on human breast cancer cells and bone marrow were established using cell proliferation and colony formation assays, and the efficiency of cell kill was evaluated. Finally, the efficiency of JS1/34.5−/47− purging of breast cancer cells was examined in cocultures of breast cancer cells with bone marrow as well as bone marrow samples from high-risk breast cancer patients. Results: We show effective killing of human breast cancer cell lines with the JS1/34.5−/47− virus. Furthermore, we show that treatment with JS1/34.5−/47− can significantly inhibit the growth of breast cancer cell lines without affecting cocultured mononuclear hematopoietic cells. Finally, we have found that the virus is effective in destroying disseminated tumors cells in bone marrow taken from breast cancer patients, without affecting the hematopoietic contents in these samples. Conclusion: Collectively, our data show that the JS1/34.5−/47− virus can selectively target breast cancer cells while sparing hematopoietic cells, suggesting that JS1/34.5−/47− can be used to purge contaminating breast cancer cells from human bone marrow in the setting of autologous hematopoietic cell transplantation.

MDR1 is upregulated in many tumors. We have previously detected activation of the MDR1 upstream promoter in metastatic breast cancer cells. MDR1 overlaps with an uncharacterized gene transcribed from the opposite strand, coding for Rap2 interacting protein 9 (RPIP9). Rap2 belongs to the Ras superfamily of GTPases, whose role in breast cancer remains unknown. We developed sensitive methods for detecting and quantifying RPIP9 mRNA and used it to identify these transcripts in normal human tissues, 60 biopsies of primary breast carcinoma, in isolated epithelial cells both from the primary tumor and from associated lymph nodes, and from bone marrow biopsies of 74 breast cancer patients. RPIP9 is expressed at high levels in normal testis, brain and adrenal gland, and at very low levels in normal breast. Tumorigenic breast carcinoma cell lines expressed RPIP9, whereas MCF-10A and HBL-100 that do not form tumors in nude mice had undetectable levels of RPIP9 mRNA. RPIP9 was activated in a high proportion of breast carcinomas (61.6%; n = 60) and a significant correlation with metastatic lymph node invasion (N = 0–3 vs. N > 3, where N = number of lymph nodes invaded; p = 0.031) was found. RPIP9 mRNA could be detected in malignant epithelial cells isolated from the primary tumor and from metastasized lymph nodes as well as in the bone marrow of significantly more poor-prognosis (N > 3) than better-prognosis (N = 0–3) patients (p = 0.001). Therefore, activation of RPIP9 occurs during the malignant breast epithelial transformation and increases with progression toward an invasive phenotype. © 2005 Wiley-Liss, Inc.

Purpose: Ion channel activity is involved in several basic cellular behaviors that are integral to metastasis (e.g., proliferation, motility, secretion, and invasion), although their contribution to cancer progression has largely been ignored. The purpose of this study was to investigate voltage-gated Na+ channel (VGSC) expression and its possible role in human breast cancer. Experimental Design: Functional VGSC expression was investigated in human breast cancer cell lines by patch clamp recording. The contribution of VGSC activity to directional motility, endocytosis, and invasion was evaluated by in vitro assays. Subsequent identification of the VGSC α-subunit(s) expressed in vitro was achieved using reverse transcription-PCR, immunocytochemistry, and Western blot techniques and used to investigate VGSCα expression and its association with metastasis in vivo. Results: VGSC expression was significantly up-regulated in metastatic human breast cancer cells and tissues, and VGSC activity potentiated cellular directional motility, endocytosis, and invasion. Reverse transcription-PCR revealed that Nav1.5, in its newly identified “neonatal” splice form, was specifically associated with strong metastatic potential in vitro and breast cancer progression in vivo. An antibody specific for this form confirmed up-regulation of neonatal Nav1.5 protein in breast cancer cells and tissues. Furthermore, a strong correlation was found between neonatal Nav1.5 expression and clinically assessed lymph node metastasis. Conclusions: Up-regulation of neonatal Nav1.5 occurs as an integral part of the metastatic process in human breast cancer and could serve both as a novel marker of the metastatic phenotype and a therapeutic target.

Tamoxifen has contributed to a dramatic reduction in breast cancer mortality and recent results indicate that aromatase inhibitors may further improve survival in some patients. Nevertheless, a substantial proportion of patients become resistant to treatment. To date, with the exception of estrogen receptor (ER) determination by ligand binding or immunohistochemical techniques, there has been no way of predicting which of several therapies is indicated in particular patients. We describe a novel assay using the adenoviral gene delivery system to assess ER function in breast cancer cells derived directly from patients. The purification and short-term culture of these cells has been recently described by our laboratory. Adenovirus containing an estrogen-regulated β-galactosidase reporter gene (ERE-lacZ) was constructed and used to test ER activity in breast cancer cells derived from 18 patients with primary and 16 patients with metastatic cancer, under varying treatment schedules. The adenoviral assay enabled ER activity to be readily determined in purified cells from primary breast cancers and secondary sites. Breast cancers cells could be categorized on the basis of ER activity in the absence of ligand, the presence of estrogen or anti-estrogens. In primary breast cancers, our results correlated with ER determination by immunohistochemistry in 78% of cases. In patients who had become resistant to tamoxifen, however, we found some in whom reporter activity was stimulated by tamoxifen and others whose tumors were either still estrogen responsive or completely unresponsive, irrespective of the original ER content. Our findings indicate that this reporter assay could be useful in decisions regarding use of adjuvant endocrine therapies in breast cancer. © 2003 Wiley-Liss, Inc.

Objective: This study was designed to determine the maximum tolerable dose (MTD) of gemcitabine plus docetaxel, both given on a weekly schedule, in patients with pretreated metastatic breast cancer (MBC). Methods: Heavily pretreated patients with MBC, aged 18–75 years with World Health Organization performance status of 0–2 were enrolled. Three escalating weekly doses of docetaxel (30, 35 and 40 mg/m2) followed by a weekly fixed dose of gemcitabine, 800 mg/m2, were administered on days 1, 8 and 15 of a 28-day cycle. Dose-limiting toxicity (DLT) included grade >3 hematologic toxicity and grade >2 stomatitis, asthenia, diarrhea or organ-specific toxicity (except alopecia). Dose escalation was stopped if ≧3 of 5 patients at any dose level experienced DLT. Results: Eighteen patients (median age 56 years) received a mean of 4.1 (range 1–6) cycles. Asthenia, stomatitis and leukopenia were the main DLTs. One of 5 patients had DLT at dose level 1 and 2 of 5 patients at dose level 2. At dose level 3, 3 of 5 patients had DLTs. Three additional patients treated at dose level 3 confirmed that the MTD had been reached. Therefore, the recommended docetaxel dose in combination with gemcitabine 800 mg/m2 for phase II studies was established at the next lower dose, 35 mg/m2. Of 12 evaluable patients, 7 (58%) achieved an objective response. Conclusions: Gemcitabine 800 mg/m2 plus docetaxel 35 mg/m2 on days 1, 8 and 15 of a 28-day cycle is a safe regimen which shows activity in heavily pretreated patients with MBC. Further phase II investigations with this combination are now warranted.