Targeted intracellular delivery of dimeric STINGa by two pHLIP peptides for treatment of solid tumors.

Introduction: We have developed a delivery approach that uses two pHLIP peptides that collaborate in the targeted intracellular delivery of a single payload, dimeric STINGa (dMSA). Methods: dMSA was conjugated with two pHLIP peptides via S-S cleavable self-immolating linkers to form 2pHLIP-dMSA. Results: Biophysical studies were carried out to confirm pH-triggered interactions of the 2pHLIP-dMSA with membrane lipid bilayers. The kinetics of linker self-immolation and dMSA release, the pharmacokinetics, the binding to plasma proteins, the stability of the agent in plasma, the targeting and resulting cytokine activation in tumors, and the biodistribution of the construct was investigated. This is the first study demonstrating that combining the energy of the membrane-associated folding of two pHLIPs can be utilized to enhance the targeted intracellular delivery of large therapeutic cargo payloads. Discussion: Linking two pHLIPs to the cargo extends blood half-life, and targeted delivery of dimeric STINGa induces tumor eradication and the development of robust anti-cancer immunity.

pHLIP targeted intracellular delivery of calicheamicin.

Calicheamicin is a potent, cell-cycle independent enediyne antibiotic that binds and cleaves DNA. Toxicity has led to its use in a targeted form, as an antibody-drug conjugate approved for the treatment of liquid tumors. We used a reduced calicheamicin to conjugate it to a single cysteine residue at the membrane-inserting end of a pH Low Insertion Peptide (pHLIP) that targets imaging and therapeutic agents to tumors. The cytoplasmic reduction of the disulfide releases the calicheamicin, and activation, DNA binding, and strand scission ensue. We studied the interaction of pHLIP-calicheamicin with liposomal and cellular membranes and demonstrated that the agent exhibits cytotoxic activity both in highly proliferative cancer cells and in non-proliferative immune cells, such as polarized M2 macrophages. In vivo, the agent was effective in inhibiting tumor growth in mice with no signs of toxicity. Biodistribution studies confirmed tumor targeting with no accumulation of the agent in organs and tissues. The agent was found within the tumor mass and tumor-stroma interface. Treatment of tumors led to the depletion of CD206+ M2- tumor-associated macrophages within the tumor core. pHLIP-calicheamicin could be pursued as an effective therapeutic for the treatment of solid tumors.

Treatment and Prognosis of Patients with Lung Cancer and Combined Interstitial Lung Disease.

BACKGROUND: Interstitial lung disease (ILD) is associated with a higher lung cancer (LC) risk and may impact cancer's clinical characteristics, treatment strategies, and outcomes. This impact's extent is unclear, particularly in Caucasians. METHODS: In this retrospective observational study, we reviewed the files of all LC patients diagnosed in a 38-month period. Expert radiologists reviewed the computed tomography scans performed at diagnosis. Patients with LC and ILD (n = 29, 7%) were compared to those without ILD (n = 363, 93%) for population and cancer characteristics, treatments, and clinical outcomes. RESULTS: Patients with LC and ILD were older (73 ± 8 vs. 65 ± 11 years; p < 0.001). There was no significant difference in LC histology, localization, stage, or treatment modalities. The respiratory complication rate after cancer treatment was significantly higher in the ILD group (39% vs. 6%; p < 0.01). Overall survival rates were similar at 12 (52% vs. 59%; p = 0.48) and 24 months (41% vs. 45%; p = 0.64) but poorer in the ILD group at 36 months, although not statistically significant (9% vs. 39%; p = 0.06). The ILD group had a higher probability of death (hazard ratio (HR) = 1.49 [0.96;2.27]), but this was not statistically significant (p = 0.06). In a Cox regression model, patients with ILD treated surgically had a significantly higher mortality risk (HR = 2.37 [1.1;5.09]; p = 0.03). CONCLUSIONS: Patients with combined LC and ILD have worse clinical outcomes even when similar treatment modalities are offered.

Systemic Inflammation/Nutritional Status Scores Are Prognostic but Not Predictive in Metastatic Non-Small-Cell Lung Cancer Treated with First-Line Immune Checkpoint Inhibitors.

Biomarkers of systemic inflammation/nutritional status have been associated with outcomes in advanced-stage non-small-cell lung cancer (NSCLC) treated with immune checkpoint inhibitors (ICIs). However, most of them were not tested in cohorts of patients treated with ICIs in combination with chemotherapy (CT) (ICI + CT) or with CT alone, making it impossible to discriminate a predictive from a prognostic effect. We conducted a single-center retrospective study to search for associations between various baseline biomarkers/scores that reflected the systemic inflammation/nutritional status (Lung Immune Prognostic Index, Modified Lung Immune Prognostic Index, Scottish Inflammatory Prognostic Score, Advanced Lung Cancer Inflammation Index, EPSILoN, Prognostic Nutritional Index, Systemic Immune-Inflammation Index, Gustave Roussy Immune Score, Royal Marsden Hospital Prognostic Score, Lung Immuno-oncology Prognostic Score 3, Lung Immuno-oncology Prognostic Score 4, score published by Holtzman et al., and Glasgow Prognostic Score) and outcomes in metastatic NSCLC treated in a first-line setting either with ICI in monotherapy (cohort 1; n = 75), ICI + CT (cohort 2; n = 56), or CT alone (cohort 3; n = 221). In the three cohorts, the biomarkers/scores were moderately associated with overall survival (OS) and progression-free survival (PFS). Their prognostic performance was relatively poor, with a maximum c-index of 0.66. None of them was specific to ICIs and could help to choose the best treatment modality. The systemic inflammation/nutritional status, associated with outcomes independently of the treatment, is therefore prognostic but not predictive in metastatic NSCLC.

Comparison of Retinal Imaging Techniques in Individuals with Pulmonary Artery Hypertension Using Vessel Generation Analysis.

(1) Background: Retinal vascular imaging plays an essential role in diagnosing and managing chronic diseases such as diabetic retinopathy, sickle cell retinopathy, and systemic hypertension. Previously, we have shown that individuals with pulmonary arterial hypertension (PAH), a rare disorder, exhibit unique retinal vascular changes as seen using fluorescein angiography (FA) and that these changes correlate with PAH severity. This study aimed to determine if color fundus (CF) imaging could garner identical retinal information as previously seen using FA images in individuals with PAH. (2) Methods: VESGEN, computer software which provides detailed vascular patterns, was used to compare manual segmentations of FA to CF imaging in PAH subjects (n = 9) followed by deep learning (DL) processing of CF imaging to increase the speed of analysis and facilitate a noninvasive clinical translation. (3) Results: When manual segmentation of FA and CF images were compared using VESGEN analysis, both showed identical tortuosity and vessel area density measures. This remained true even when separating images based on arterial trees only. However, this was not observed with microvessels. DL segmentation when compared to manual segmentation of CF images showed similarities in vascular structure as defined by fractal dimension. Similarities were lost for tortuosity and vessel area density when comparing manual CF imaging to DL imaging. (4) Conclusions: Noninvasive imaging such as CF can be used with VESGEN to provide an accurate and safe assessment of retinal vascular changes in individuals with PAH. In addition to providing insight into possible future clinical translational use.

Targeting Bladder Urothelial Carcinoma with pHLIP-ICG and Inhibition of Urothelial Cancer Cell Proliferation by pHLIP-amanitin.

Acidity is a useful biomarker for the targeting of metabolically active-cells in tumors. pH Low Insertion Peptides (pHLIPs) sense the pH at the surfaces of tumor cells and can facilitate intracellular delivery of cell-permeable and cell-impermeable cargo molecules. In this study we have shown the targeting of malignant lesions in human bladders by fluorescent pHLIP agents, intracellular delivery of amanitin toxin by pHLIP for the inhibition of urothelial cancer cell proliferation, and enhanced potency of pHLIP-amanitin for cancer cells with 17p loss, a mutation frequently present in urothelial cancers. Twenty-eight ex-vivo bladder specimens, from patients undergoing robotic assisted laparoscopic radical cystectomy for bladder cancer, were treated via intravesical incubation for 15-60 minutes with pHLIP conjugated to indocyanine green (ICG) or IR-800 near infrared fluorescent (NIRF) dyes at concentrations of 4-8 µM. White light cystoscopy identified 47/58 (81%) and NIRF pHLIP cystoscopy identified 57/58 (98.3%) of malignant lesions of different subtypes and stages selected for histopathological processing. pHLIP NIRF imaging improved diagnosis by 17.3% (p < 0.05). All carcinoma-in-situ cases missed by white light cystoscopy were targeted by pHLIP agents and were diagnosed by NIRF imaging. We also investigated the interactions of pHLIP-amanitin with urothelial cancer cells of different grades. pHLIP-amanitin produced concentration- and pH-dependent inhibition of the proliferation of urothelial cancer cells treated for 2 hrs at concentrations up to 4 µM. A 3-4x enhanced cytotoxicity of pHLIP-amanitin was observed for cells with a 17p loss after 2 hrs of treatment at pH6. Potentially, pHLIP technology may improve the management of urothelial cancers, including imaging of malignant lesions using pHLIP-ICG for diagnosis and surgery, and the use of pHLIP-amanitin for treatment of superficial bladder cancers via intravesical instillation.

D-dimer Testing in Pulmonary Embolism with a Focus on Potential Pitfalls: A Narrative Review.

D-dimer is a multifaceted biomarker of concomitant activation of coagulation and fibrinolysis, which is routinely used for ruling out pulmonary embolism (PE) and/or deep vein thrombosis (DVT) combined with a clinical pretest probability assessment. The intended use of the tests depends largely on the assay used, and local guidance should be applied. D-dimer testing may suffer from diagnostic errors occurring throughout the pre-analytical, analytical, and post-analytical phases of the testing process. This review aims to provide an overview of D-dimer testing and its value in diagnosing PE and discusses the variables that may impact the quality of its laboratory assessment.

Eradication of tumors and development of anti-cancer immunity using STINGa targeted by pHLIP.

Despite significant progress in the development of novel STING agonists (STINGa), applications appear to be challenged by the low efficiency and poor selectivity of these agents. A pH Low Insertion Peptide (pHLIP) extends the lifetime of a STINGa in the blood and targets it to acidic cancer-associated fibroblasts (CAFs), tumor-associated macrophages (TAMs), myeloid derived suppressor cells (mMDSCs) and dendritic cells (DCs). CAFs constitute 25% of all live cells within CT26 tumors, and M2-type TAMs and mMDSCs are the most abundant among the immune cells. The resulting activation of cytokines within the tumor microenvironment (TME) triggers the eradication of small (100 mm3) and large (400-700 mm3) CT26 tumors in mice after a single dose of pHLIP-STINGa. The tumor stroma was destroyed (the number of CAFs was reduced by 98%), intratumoral hemorrhage developed, and the level of acidity within the TME was reduced. Further, no tumors developed in 20 out of 25 tumor-free mice re-challenged by an additional injection of cancer cells. The therapeutic effect on CT26 tumors was insignificant in nude mice, lacking T-cells. Thus, targeted delivery of STINGa to tumor stroma and TAMs induces activation of signaling, potentially resulting in the recruitment and infiltration of T-cells, which gain access to the tumor core. The cytotoxic activity of T-cells is not impaired by an acidic environment and immune memory is developed.

Case report: BRAF A598-T599insV mutation as a potential resistance mechanism to alectinib in ALK-rearranged lung adenocarcinoma.

Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) have improved the prognosis of advanced-stage non-small cell lung cancer (NSCLC) with ALK rearrangement, but resistance mechanisms limit their efficacy. We describe the case of a 63-year-old man with a stage cIVA ALK-rearranged lung adenocarcinoma who developed a BRAF A598-T599insV mutation as a potential resistance mechanism to alectinib, a second-generation ALK TKI. He was treated with an association of BRAF and MEK inhibitors but death occurred two months after treatment initiation in a context of tumor progression and toxicity. Based on this first report of BRAF A598-T599insV mutation occurring in lung cancer, we discuss resistance mechanisms to ALK TKIs, implications of BRAF mutation in NSCLC, and BRAF A598-T599insV mutation in other cancers.

pHLIP Peptides Target Acidity in Activated Macrophages.

PURPOSE: Acidity can be a useful alternative biomarker for the targeting of metabolically active cells in certain diseased tissues, as in acute inflammation or aggressive tumors. We investigated the targeting of activated macrophages by pH low insertion peptides (pHLIPs), an established technology for targeting cell-surface acidity. PROCEDURES: The uptake of fluorescent pHLIPs by activated macrophages was studied in cell cultures, in a mouse model of lung inflammation, and in a mouse tumor model. Fluorescence microscopy, whole-body and organ imaging, immunohistochemistry, and FACS analysis were employed. RESULTS: We find that cultured, activated macrophages readily internalize pHLIPs. The uptake is higher in glycolytic macrophages activated by LPS and INF-γ compared to macrophages activated by IL-4/IL-13. Fluorescent pHLIPs target LPS-induced lung inflammation in mice. In addition to marking cancer cells within the tumor microenvironment, fluorescent pHLIPs target CD45+, CD11b+, F4/80+, and CD206+ tumor-associated macrophages with no significant targeting of other immune cells. Also, fluorescent pHLIPs target CD206-positive cells found in the inguinal lymph nodes of animals inoculated with breast cancer cells in mammary fat pads. CONCLUSIONS: pHLIP peptides sense low cell surface pH, which triggers their insertion into the cell membrane. Unlike cancerous cells, activated macrophages do not retain inserted pHLIPs on their surfaces, instead their highly active membrane recycling moves the pHLIPs into endosomes. Targeting activated macrophages in diseased tissues may enable clinical visualization and therapeutic opportunities.

Abdominal CT findings in Puumala hantavirus-infected patients.

BACKGROUND: Previous clinical studies have reported abdominal findings on ultrasonography or MRI in Puumala hantavirus-infected patients. PURPOSE: To determine if abdominal computed tomography (CT) can lead to a diagnosis of Puumala virus infection in the presence of a suggestive clinical picture. MATERIAL AND METHODS: CT findings were studied retrospectively in 30 patients who presented to the emergency department of two (Belgian) hospitals with serologically confirmed Puumala hantavirus infection. RESULTS: The most frequent finding was perirenal fascial thickening (90%), followed by perirenal fat stranding (87%). Retroperitoneal fat stranding was found in 19 patients (64%) in the perivesical spaces along the fascia of the external iliac vessels with or without involvement of the presacral fat. Half of the patients had pelvic ascites, and pleural fluid was found in 7 of them. The right and left mean pole-to-pole kidney's lengths were respectively 125.7 mm and 127.8 mm in 28 patients. CONCLUSION: Retroperitoneal fat stranding, perirenal fascial thickening and/or perirenal fat stranding were found in most patients with acute Puumala virus infection who have undergone CT. Although nonspecific, these findings may help to suggest Puumala hantavirus infection in the right clinical settings.

Cardiac CT findings in patients with family history of premature CAD: an observational study.

INTRODUCTION: A family history of premature CAD may promote enhanced development of coronary atherosclerosis in a sibling population. Baseline CV algorithms may underestimate the risk of coronary incidents in individuals at familial risk. Cardiac CT provides high diagnostic performance for the detection of coronary plaques. There is little data on the use of this technology in the initial diagnostic approach of these patients. The prognostic value of early detection of coronary plaques by cardiac CT remains unknown in this population. OBJECTIVES: The study aimed to estimate the global CV risk and the pre-test probability of CAD in patients with a family history of premature CAD. We investigated the potential role of cardiac CT imaging in the assessment of coronary risk in patients from high-risk families. We sought to remind the 2019 ESC guidelines for screening for CAD in asymptomatic subjects. METHODS: Fifty siblings of patients with premature CAD were investigated. The pre-test probability of CAD was determined with the Clinical Model of the CAD consortium. The risk of CV disease was calculated and compared with three different risk algorithms (SCORE, FRS, PROCAM). All patients underwent cardiac CT with both non-contrast and contrast imaging. Coronary artery calcium (CAC) scoring was calculated and CT angiograms were analyzed. Patients with suspected CT obstructive CAD underwent coronary angiography. Clinical outcomes in terms of treatment were analyzed. RESULTS: The pre-test probability of CAD was low: CAD consortium <10% in 60%, SCORE <5% in 100%, FRS <10% in 88%, CAC scoring <100% in 68%. However, PROCAM was <10% in 16 cases (32%). Only 12 patients (24%) presented normal CCTA findings. In patients with abnormal CCTA findings (n = 38), PROCAM was higher than FRS in 20 patients (53%). Coronary angiography was performed in 31 cases (62%) for suspected CT obstructive CAD. Most patients presented no significant lesions (55%). Revascularization was performed in 8 patients (16%), 6 of them (75%) presented CAC scoring <100, 4 of them (50%) presented CAC scoring <400. After investigation, lipid-lowering therapy was enhanced by 66%. CONCLUSION: Coronary atherosclerotic-phenotyping using cardiac CT may provide discriminatory information allowing earlier identification of patients at familial risk of premature CAD. This diagnostic workup strategy may help to guide and improve the management of these patients. However, there is a paucity of data concerning the prognostic significance of this technology in relatives at familial risk of premature CAD. Therefore, further randomized controlled trials are needed to assess the incremental risk-predictive value of this approach in this population.

Tumor treatment by pHLIP-targeted antigen delivery.

Targeted antigen delivery allows activation of the immune system to kill cancer cells. Here we report the targeted delivery of various epitopes, including a peptide, a small molecule, and a sugar, to tumors by pH Low Insertion Peptides (pHLIPs), which respond to surface acidity and insert to span the membranes of metabolically activated cancer and immune cells within tumors. Epitopes linked to the extracellular ends of pH Low Insertion Peptide peptides were positioned at the surfaces of tumor cells and were recognized by corresponding anti-epitope antibodies. Special attention was devoted to the targeted delivery of the nine residue HA peptide epitope from the Flu virus hemagglutinin. The HA sequence is not present in the human genome, and immunity is readily developed during viral infection or immunization with KLH-HA supplemented with adjuvants. We tested and refined a series of double-headed HA-pHLIP agents, where two HA epitopes were linked to a single pH Low Insertion Peptide peptide via two Peg12 or Peg24 polymers, which enable HA epitopes to engage both antibody binding sites. HA-epitopes positioned at the surfaces of tumor cells remain exposed to the extracellular space for 24-48 h and are then internalized. Different vaccination schemes and various adjuvants, including analogs of FDA approved adjuvants, were tested in mice and resulted in a high titer of anti-HA antibodies. Anti-HA antibody binds HA-pHLIP in blood and travels as a complex leading to significant tumor targeting with no accumulation in organs and to hepatic clearance. HA-pHLIP agents induced regression of 4T1 triple negative breast tumor and B16F10 MHC-I negative melanoma tumors in immunized mice. The therapeutic efficacy potentially is limited by the drop of the level of anti-HA antibodies in the blood to background level after three injections of HA-pHLIP. We hypothesize that additional boosts would be required to keep a high titer of anti-HA antibodies to enhance efficacy. pH Low Insertion Peptide-targeted antigen therapy may provide an opportunity to treat tumors unresponsive to T cell based therapies, having a small number of neo-antigens, or deficient in MHC-I presentation at the surfaces of cancer cells either alone or in combination with other approaches.

Subgaleal Lipoma: Imaging Findings.

Subgaleal lipoma is a benign tumor of adipose tissue. It should be suspected when a semi-spherical avascular mass with well-defined margins, iso- or hyperechoic in most cases, with thin internal echogenic lines parallel to the long axis of the tumor, is observed between the galea aponeurosis and periosteum of the cranial bone. We report a series of cases of three patients who underwent surgical lesion excision and whose histopathological examination findings confirmed the diagnosis of lipoma. MAIN TEACHING POINT: The presence of long continuous echogenic lines within a lens-shaped soft tissue mass located beneath the galea aponeurosis may suggest the diagnosis of subgaleal lipoma.

Presacral Myelolipoma: The Usefulness of a 99mTc-Albumin Nanocolloid Scintigraphy.

Myelolipoma is a rare mesenchymal tumor consisting of adipose tissue and hematopoietic cells. Found usually in the adrenal region, however, few cases have been reported in extra-adrenal regions, most frequently in the presacral region. It is important to recognize such tumor, as it can attain massive size and causes pressure symptoms, and needs to be differentiated from malignant tumors, including liposarcomas. Although CT and MRI can suggest a diagnosis of myelolipoma, these are not conclusive. The hematopoietic cells are enhanced by a Tc-albumin nanocolloid scintigraphy and help to distinguish myolipoma from other entities.

pHLIP ICG for delineation of tumors and blood flow during fluorescence-guided surgery.

Fluorescence imaging has seen enduring use in blood flow visualization and is now finding a new range of applications in image-guided surgery. In this paper, we report a translational study of a new fluorescent agent for use in surgery, pHLIP ICG, where ICG (indocyanine green) is a surgical fluorescent dye used widely for imaging blood flow. We studied pHLIP ICG interaction with the cell membrane lipid bilayer, the pharmacology and toxicology in vitro and in vivo (mice and dogs), and the biodistribution and clearance of pHLIP ICG in mice. The pHLIP ICG tumor targeting and imaging efficacy studies were carried out in several murine and human mouse tumor models. Blood vessels were imaged in mice and pigs. Clinical Stryker imaging instruments for endoscopy and open surgery were used in the study. Intravenously administered pHLIP ICG exhibits a multi-hour circulation half-life, offering protracted delineation of vasculature. As it clears from the blood, pHLIP ICG targets tumors and tumor stroma, marking them for surgical removal. pHLIP ICG is non-toxic, marks blood flow for hours after injection, and effectively delineates tumors for improved resection on the day after administration.

Early Chest Imaging in Patients with Puumala Hantavirus Infection.

PURPOSE: To describe early chest imaging abnormalities in patients with acute Puumala virus infection. MATERIALS AND METHODS: This retrospective study (2005-2017) comprised 64 patients who were admitted to the emergency department of a Belgian hospital. These patients were diagnosed with serologically confirmed acute Puumala virus infection and had at least one chest X-ray (CRX). Imaging studies were evaluated by two experienced chest radiologists reaching agreement by consensus, and abnormalities were reported according to the Fleischner Society glossary of terms for thoracic imaging. When a patient underwent multiple CRX, only the findings of the first were recorded. Six patients underwent chest high-resolution computed tomography (HRCT). RESULTS: CRX showed abnormal findings in 33 patients (51.5%). Most common findings were linear atelectasis (29.7%) and small pleural effusion (20.3%). HRCT showed interlobular septal thickening in four patients and crazy-paving pattern with consolidations in one patient with adult respiratory distress syndrome. CONCLUSIONS: Early CRX commonly showed linear atelectasis and small pleural effusion in Puumala virus infected patients above 30 years of age. Chest HRCT most frequently showed atelectasis and smooth interlobular septal thickening. While uncommon, early and severe pulmonary involvement can be associated with Puumala virus infection, albeit these findings are not specific.

Systematic scrotum ultrasound in male infertility: what to look for.

Ultrasound is a useful, cost-effective and minimally invasive tool that can be used in the workup of male infertility. Patient history, semen analysis and hormonal results often precede ultrasound examination as a part of the workup of male factor infertility.In our article, we advocate the added value of a systematic approach of the scrotal ultrasound. We propose a checklist for the complete analysis of testicular and paratesticular structures, useful in everyday practice for both clinicians and radiologists, highlighting what can be expected of and what should be found in the radiologist's report.

Puumala hantavirus: an imaging review.

Puumala virus (PUUV) is the most common hantavirus in Europe. It is known to cause nephropathia epidemica, which is considered a mild type of hemorrhagic fever with renal syndrome. However, it does not only involve the kidneys and is rarely accompanied by symptomatic hemorrhage. We review the imaging abnormalities caused by PUUV infection, from head to pelvis, emphasizing the broad spectrum of possible findings and bringing further support to a previously suggested denomination "Hantavirus disease" that would encompass all clinical manifestations. Although non-specific, knowledge of radiological appearances is useful to support clinically suspected PUUV infection, before confirmation by serology.