Protocol for phage matching, treatment, and monitoring for compassionate bacteriophage use in non-resolving infections.

Phage therapy has re-emerged as a promising treatment for non-resolving infections. Given the lack of approved phage treatments, there is a need to establish a compassionate use pipeline. Here, we present a protocol for phage matching, treatment, and monitoring for compassionate bacteriophage use in non-resolving infections. We describe steps for consultation and request implementation, evaluating and comparing different aspects of phage activity, and phage production. We then detail procedures for multidisciplinary meetings, ethics approvals, phage therapy, and follow-up. For complete details on the use and execution of this protocol, please refer to Onallah et al.1,2.

Pseudomonas aeruginosa ventricular assist device infections: findings from ineffective phage therapies in five cases.

Left ventricular assist devices (LVAD) are increasingly used for management of heart failure; infection remains a frequent complication. Phage therapy has been successful in a variety of antibiotic refractory infections and is of interest in treating LVAD infections. We performed a retrospective review of four patients that underwent five separate courses of intravenous (IV) phage therapy with concomitant antibiotic for treatment of endovascular Pseudomonas aeruginosa LVAD infection. We assessed phage susceptibility, bacterial strain sequencing, serum neutralization, biofilm activity, and shelf-life of phage preparations. Five treatments of one to four wild-type virulent phage(s) were administered for 14-51 days after informed consent and regulatory approval. There was no successful outcome. Breakthrough bacteremia occurred in four of five treatments. Two patients died from the underlying infection. We noted a variable decline in phage susceptibility following three of five treatments, four of four tested developed serum neutralization, and prophage presence was confirmed in isolates of two tested patients. Two phage preparations showed an initial titer drop. Phage biofilm activity was confirmed in two. Phage susceptibility alone was not predictive of clinical efficacy in P. aeruginosa endovascular LVAD infection. IV phage was associated with serum neutralization in most cases though lack of clinical effect may be multifactorial including presence of multiple bacterial isolates with varying phage susceptibility, presence of prophages, decline in phage titers, and possible lack of biofilm activity. Breakthrough bacteremia occurred frequently (while the organism remained susceptible to administered phage) and is an important safety consideration.

Towards Standardization of Phage Susceptibility Testing: The Israeli Phage Therapy Center "Clinical Phage Microbiology"-A Pipeline Proposal.

Using phages as salvage therapy for nonhealing infections is gaining recognition as a viable solution for patients with such infections. The escalating issue of antibiotic resistance further emphasizes the significance of using phages in treating bacterial infections, encompassing compassionate-use scenarios and clinical trials. Given the high specificity of phages, selecting the suitable phage(s) targeting the causative bacteria becomes critical for achieving treatment success. However, in contrast to conventional antibiotics, where susceptibility-testing procedures were well established for phage therapy, there is a lack of standard frameworks for matching phages from a panel to target bacterial strains and assessing their interactions with antibiotics or other agents. This review discusses and compares published methods for clinical phage microbiology, also known as phage susceptibility testing, and proposes guidelines for establishing a standard pipeline based on our findings over the past 5 years of phage therapy at the Israeli Phage Therapy Center.

Refractory Pseudomonas aeruginosa infections treated with phage PASA16: A compassionate use case series.

BACKGROUND: A growing number of compassionate phage therapy cases were reported in the last decade, with a limited number of clinical trials conducted and few unsuccessful clinical trials reported. There is only a little evidence on the role of phages in refractory infections. Our objective here was to present the largest compassionate-use single-organism/phage case series in 16 patients with non-resolving Pseudomonas aeruginosa infections. METHODS: We summarized clinical phage microbiology susceptibility data, administration protocol, clinical data, and outcomes of all cases treated with PASA16 phage. In all intravenous phage administrations, PASA16 phage was manufactured and provided pro bono by Adaptive Phage Therapeutics. PASA16 was administered intravenously, locally to infection site, or by topical use to 16 patients, with data available for 15 patients, mainly with osteoarticular and foreign-device-associated infections. FINDINGS: A few minor side effects were noted, including elevated liver function enzymes and a transient reduction in white blood cell count. Good clinical outcome was documented in 13 out of 15 patients (86.6%). Two clinical failures were reported. The minimum therapy duration was 8 days with a once- to twice-daily regimen. CONCLUSIONS: PASA16 with antibiotics was found to be relatively successful in patients for whom traditional treatment approaches have failed previously. Such pre-phase-1 cohorts can outline potential clinical protocols and facilitate the design of future trials. FUNDING: The study was funded in part by The Israeli Science Foundation IPMP (ISF_1349/20), Rosetrees Trust (A2232), United States-Israel Binational Science Foundation (2017123), and the Milgrom Family Support Program.

Compassionate Use of Bacteriophages for Failed Persistent Infections During the First 5 Years of the Israeli Phage Therapy Center.

The use of bacteriophages (phages) is reemerging as a potential treatment option for antibiotic-resistant or nonresolving bacterial infections. Phages are bacteria-specific viruses that may serve as a personalized therapeutic option with minimal collateral damage to the patient or the microbiome. In 2018 we established the Israeli Phage Therapy Center (IPTC) as a shared initiative of the Hadassah Medical Center and the Hebrew University of Jerusalem, aiming to conduct all of the steps required for phage-based solutions, from phage isolation and characterization to treatments, for nonresolving bacterial infections. So far, a total of 159 requests for phage therapy arrived to the IPTC; 145 of them were from Israel and the rest from other countries. This number of registered requests is growing annually. Multidrug-resistant bacteria accounted for 38% of all phage requests. Respiratory and bone infections were the most prevalent among clinical indications and accounted for 51% of the requests. To date, 20 phage therapy courses were given to 18 patients by the IPTC. In 77.7% (n = 14) of the cases, a favorable clinical outcome of infection remission or recovery was seen. Clearly, establishing an Israeli phage center has led to an increased demand for compassionate use of phages with favorable outcomes for many previously failed infections. As clinical trials are still lacking, publishing patient data from cohort studies is pertinent to establish clinical indications, protocols, and success and failure rates. Last, workflow processes and bottlenecks should be shared to enable faster availability and authorization of phages for clinical use.

Exosome Secretion and Epithelial-Mesenchymal Transition in Ovarian Cancer Are Regulated by Phospholipase D.

Phospholipase D (PLD) isoenzymes participate in a variety of cellular functions that are mostly attributed to phosphatidic acid (PA) synthesis. Dysregulation of PLD regulates tumor progression and metastasis, yet little is known about the underlying mechanism. We previously reported on the expression and clinical role of the PLD isoenzymes PLD1 and PLD2 in tubo-ovarian high-grade serous carcinoma (HGSC). In the present study, we investigated the biological function of PLD1 and PLD2 using the OVCAR-3 and OVCAR-8 HGSC cell lines. KO cell lines for both PLDs were generated using CRISPR/CAS9 technology and assayed for exosome secretion, spheroid formation, migration, invasion and expression of molecules involved in epithelial-mesenchymal transition (EMT) and intracellular signaling. Significant differences between PLD1 and PLD2 KO cells and controls were observed for all the above parameters, supporting an important role for PLD in regulating migration, invasion, metastasis and EMT.

Diverse Effects of Lysophosphatidic Acid Receptors on Ovarian Cancer Signaling Pathways.

Lysophosphatidic acid (LPA) is a bioactive phospholipid with mitogenic and growth factor-like activities affecting cell invasion, cancer progression, and resistance. It is produced mainly by autotaxin and acts on six G-protein-coupled receptors, LPAR1-6. LPA has recently been implicated as a growth factor present in ascites of ovarian cancer patients. However, mitogenic pathways stimulated by LPA via its receptors may involve any novel, thus far uncharacterized, signaling pathway(s). Here we show that three LPA receptors are involved in tumor progression by activation of both the AKT and ERK signaling pathways. CRISPR-edited LPAR2 and LPAR3 knockouts have opposing effects on ERK activation, whereas LPAR6 is involved in the activation of AKT, affecting cell migration and invasion. Our study identifies specific molecular machinery triggered by LPA and its receptors that modulates tumor cells and can serve as therapeutic target in this malignancy.

SOX2 and SOX9 are markers of clinically aggressive disease in metastatic high-grade serous carcinoma.

OBJECTIVE: The aim of this study was to analyze the expression, biological role and clinical relevance of cancer stem cell markers in high-grade serous carcinoma (HGSC). METHODS: mRNA expression by qRT-PCR of NANOG, OCT4, SOX2, SOX4, SOX9, LIN28A and LIN28B was analyzed in 134 HGSC specimens (84 effusions, 50 surgical specimens). Nanog, OCT3/4, SOX2 and SOX9 protein expression by immunohistochemistry was analyzed in 52 HGSC effusions. Nanog protein expression in exosomes from 80 HGSC effusions was studied by Western Blotting. OVCAR3 cells underwent CRISPR/Cas9 Nanog knockout (KO), and the effect of Nanog KO on migration, invasion, proliferation and proteolytic activity was analyzed in OVCAR3 and OVCAR8 cells. RESULTS: OCT4 mRNA was overexpressed in effusions compared to solid specimens (p = 0.046), whereas SOX9 was overexpressed in the ovarian tumors compared to effusions and solid metastases (p = 0.003). Higher SOX2 and SOX9 expression was associated with primary (intrinsic) chemoresistance (p = 0.009 and p = 0.02, respectively). Higher SOX9 levels were associated with shorter overall survival in univariate (p = 0.04) and multivariate (p = 0.049) analysis. OCT3/4, SOX2 and SOX9 proteins were found in HGSC cells, whereas Nanog was detected only in exosomes. Higher SOX2 protein expression was associated with shorter overall survival in univariate analysis (p = 0.049). OVCAR cells exposed to OVCAR3 NANOG KO exosomes had reduced migration, invasion and MMP9 activity. CONCLUSIONS: SOX2 and SOX9 mRNA levels in HGSC effusions may be markers of clinically aggressive disease. Nanog is secreted in HGSC exosomes in effusions and modulates tumor-promoting cellular processes in vitro.

Activity and clinical relevance of autotaxin and lysophosphatidic acid pathways in high-grade serous carcinoma.

The aim of this study was to analyze the expression, biological role and clinical relevance of autotaxin (ATX), the enzyme synthetizing lysophosphatidic acid (LPA), and LPA receptors (LPAR) in high-grade serous carcinoma (HGSC). mRNA expression by qRT-PCR of LPAR1-6 was analyzed in 155 HGSC specimens (88 effusions, 67 solid lesions). ATX mRNA expression was analyzed in 97 specimens. ATX, ERK, and AKT protein expression was studied by Western blotting. LPAR2 mRNA was overexpressed in HGSC cells in effusions compared to solid lesions, with opposite findings for LPAR3 and LPAR6 mRNA and ATX protein. Higher LPAR1 levels were significantly related to longer overall survival (OS) in pre-chemotherapy effusions (p = 0.027). Conversely, higher expression of LPAR1, LPAR2, and LPAR5 in post-chemotherapy effusions was significantly associated with shorter OS (p = 0.037, p = 0.025 and p = 0.021, respectively) and progression-free survival (PFS) (p < 0.001, p = 0.007 and p < 0.001, respectively) in univariate survival analysis. LPAR1 mRNA expression was an independent prognosticator of OS in patients with pre-chemotherapy effusions and PFS in patients with post-chemotherapy effusions (p = 0.013 both). In conclusion, LPAR mRNA and ATX protein levels are anatomic site-dependent in HGSC and the former are informative of disease outcome.