Use of Low-Frequency Driver Mutations Detected by Cell-Free Circulating Tumor DNA to Guide Targeted Therapy in Non-Small-Cell Lung Cancer: A Multicenter Case Series.

PURPOSE: To evaluate the clinical outcome of patients with non-small-cell lung cancer treated by targeting low variant allelic frequency (VAF) driver mutations identified through cell-free DNA (cfDNA) next-generation sequencing (NGS). Detection of driver mutations in cancer is critically important in the age of targeted therapy, where both tumor-based as well as cfDNA sequencing methods have been used for therapeutic decision making. We hypothesized that VAF should not be predictive of response and that low VAF alterations detected by cfDNA NGS can respond to targeted therapy. PATIENTS AND METHODS: A multicenter retrospective case review was performed to identify patients with non-small-cell lung cancer who received targeted molecular therapy on the basis of findings of low VAF alterations in cfDNA NGS. Mutations at low VAF were defined as < 0.2% mutated cfDNA molecules in a background of wild-type cfDNA. RESULTS: One hundred seventy-two patients underwent cfDNA NGS testing. Of the 172 patients, 12 were identified as having low VAF driver alterations and were considered for targeted therapy. The median progression-free survival (PFS) for all patients was 52 weeks (range, 17 to 88 weeks). For patients with EGFR exon 19 deletion (n = 7), the median PFS was 52 weeks (range, 17 to 60.5 weeks). For patients with EML4-ALK fusions (n = 3), the median PFS was 60 weeks (range, 18 to 88 weeks). The median overall survival for all patients after diagnosis was 57.6 weeks. CONCLUSION: Targeted treatment response for driver mutations detected by cfDNA may be independent of VAF, even in relation to other higher VAF aberrations in plasma, and directly dependent on the underlying disease biology and ability to treat the patient with appropriate targeted therapy.

Targeting human C-type lectin-like molecule-1 (CLL1) with a bispecific antibody for immunotherapy of acute myeloid leukemia.

Acute myeloid leukemia (AML), which is the most common acute adult leukemia and the second most common pediatric leukemia, still has a poor prognosis. Human C-type lectin-like molecule-1 (CLL1) is a recently identified myeloid lineage restricted cell surface marker, which is overexpressed in over 90% of AML patient myeloid blasts and in leukemic stem cells. Here, we describe the synthesis of a novel bispecific antibody, αCLL1-αCD3, using the genetically encoded unnatural amino acid, p-acetylphenylalanine. The resulting αCLL1-αCD3 recruits cytotoxic T cells to CLL1 positive cells, and demonstrates potent and selective cytotoxicity against several human AML cell lines and primary AML patient derived cells in vitro. Moreover, αCLL1-αCD3 treatment completely eliminates established tumors in an U937 AML cell line xenograft model. These results validate the clinical potential of CLL1 as an AML-specific antigen for the generation of a novel immunotherapeutic for AML.

Severe disease on endoscopy and steroid use increase the risk for bowel perforation during colonoscopy in inflammatory bowel disease patients.

BACKGROUND AND AIM: Colonoscopic perforation is a rare complication. We sought to determine its risk factors in patients with inflammatory bowel disease (IBD). MATERIALS AND METHODS: The study group consisted of 19 IBD patients who had perforation secondary to diagnostic or therapeutic colonoscopy from January 2002 to October 2010. The control group consists of 76 IBD patients undergoing colonoscopy and no perforations that were matched based on indication in a 4:1 ratio to the study group. Demographic and clinical variables as well as perforation outcomes were analyzed by univariate and multivariate analyses. RESULTS: There were a total of 5295 colonoscopies done during the study period in IBD patients of which 19 patients had perforation. The prevalence of perforation in IBD patients was 0.3%. Of the 19 patients, 12 had Crohn's disease (CD) and 7 had ulcerative colitis (UC). Patients in the perforation group were more likely treated with steroids (68.4% vs. 21.1%, p<0.001) and had severe disease on endoscopy (31.6% vs. 10.1%, p=0.03) than that in the control groups. On multivariate analysis, severe disease on endoscopy (adjusted odds ratio [aOR]=3.82, 95% confidence interval [CI]=1.03-15.24) and steroid treatment (aOR=7.68; 95% CI=1.48, 39.81) were independently associated with the risk of perforation. The median length of stay in the perforation group was 10 days (range 2-23 days). There was no mortality in our study. CONCLUSIONS: There appears to be a higher risk of colonoscopy-associated perforation in IBD patients with active disease and on steroids.

Angiotensin II type 1 (AT-1) receptor inhibition partially prevents the urodynamic and detrusor changes associated with bladder outlet obstruction: a mouse model.

UNLABELLED: Study Type - Therapy (case control) Level of Evidence 3b What's known on the subject? and What does the study add? Angiotensin II is the main effector peptide in the bladder local renin-angiotensin system. This experiment demonstrates the role of this local renin-angiotensin system with respect to bladder outlet obstruction. OBJECTIVE: • To determine if treatment with an angiotensin II type 1 (AT-1) receptor antagonist, losartan, can prevent the structural and functional changes that occur in a mouse model of bladder outlet obstruction (BOO). MATERIALS AND METHODS: • Twenty-four Balb/CAN mice underwent partial urethral obstruction for 6 weeks. • Twelve mice were given oral losartan (10 mg/kg/day), and 12 were not. Six mice served as unobstructed controls, and six unobstructed mice were given oral losartan (10 mg/kg/day) to determine the effect of angiotensin II inhibition on the normal bladder. • Bladder capacity (C), detrusor pressure during voiding (Pdet) and volume at first non-voiding contraction (NVC1) as a percentage of C were recorded after 6 weeks. • Bladders were stained with haematoxylin and eosin for measurement of detrusor muscular thickness, and graded as 1 = atrophy (<100 µm thick), 2 = normal (100-200 µm thick), 3 = hypertrophy (>200 µm thick) compared with controls. RESULTS: • Compared with controls, BOO mice had greater C (153.5 ± 20.9 vs 57.5 ± 7.4 µl, P < 0.01), higher Pdet (28.8 ± 2.1 vs 12.1 ± 2.1 mm Hg), lower NVC1 (median = 24% vs 54% P= 0.03). BOO mice manifested greater bladder weight (93.2 ± 11.7 mg vs 26.8 ± 2.40 mg, P < 0.01) and greater detrusor muscle thickness (median 3 vs 2, P= 0.02). • Compared with untreated BOO mice, mice treated with losartan had greater mean C (248.8 ± 28.6 vs 153.5 ± 20.9 µL, P= 0.01), no significant change in mean Pdet (24.7 ± 1.6 vs 28.8 ± 2.1 mm Hg, P= 0.2) and a higher mean NVC1 (47% vs 24%, P= 0.02). • Treatment with losartan mediated an insignificant reduction in mean bladder weight (68.1 ± 9.1 mg vs 93.2 ± 11.7 mg, P= 0.10), but a significant reduction in detrusor muscle thickness (median 2 vs 3, P= 0.02). Losartan did not mediate any significant structural or functional changes in the unobstructed mouse bladder. CONCLUSION: • In a mouse model of BOO, treatment with an AT-1 antagonist partially prevented the urodynamic and structural changes that otherwise occur with BOO.

Vulnerability of continence structures to injury by simulated childbirth.

The goal of this study was to examine acute morphological changes, edema, muscle damage, inflammation, and hypoxia in urethral and vaginal tissues with increasing duration of vaginal distension (VD) in a rat model. Twenty-nine virgin Sprague-Dawley rats underwent VD under anesthesia with the use of a modified Foley catheter inserted into the vagina and filled with saline for 0, 1, 4, or 6 h. Control animals were anesthetized for 4 h without catheter placement. Urogenital organs were harvested after intracardiac perfusion of fixative. Tissues were embedded, sectioned, and stained with Masson's trichrome or hematoxylin and eosin stains. Regions of hypoxia were measured by hypoxyprobe-1 immunohistochemistry. Within 1 h of VD, the urethra became vertically elongated and displaced anteriorly. Edema was most prominent in the external urethral sphincter (EUS) and urethral/vaginal septum within 4 h of VD, while muscle disruption and fragmentation of the EUS occurred after 6 h. Inflammatory damage was characterized by the presence of polymorphonuclear leukocytes in vessels and tissues after 4 h of VD, with the greatest degree of infiltration occurring in the EUS. Hypoxia localized mostly to the vaginal lamina propria, urethral smooth muscle, and EUS within 4 h of VD. Increasing duration of VD caused progressively greater tissue edema, muscle damage, and morphological changes in the urethra and vagina. The EUS underwent the greatest insult, demonstrating its vulnerability to childbirth injury.

Chronic sacral nerve stimulation prevents detrusor structural and functional changes associated with bladder outlet obstruction--a rat model.

AIMS: Bladder outlet obstruction (BOO) can mediate structural and functional detrusor changes, which can lead to bothersome lower urinary tract symptoms. We investigate if sacral nerve stimulation (SNS) can prevent these structural and functional changes in a rat model of BOO. METHODS: 24 female Sprague-Dawley rats (250 gm) were divided into 4 groups: control (CTRL), BOO, SNS, and both (BOO/SNS). BOO was achieved by partially occluding the proximal urethra. SNS involved stimulating the S1-S4 dorsal roots with a unipolar S1 lead, 8 hours daily. Urodynamics were performed at baseline and after 6 weeks. Bladders were harvested, stained, and scored for detrusor hypertrophy and fibrosis (scale = 1-5). RESULTS: BOO caused an increase in mean voiding pressure (P(det) = 35 +/- 2 mmHg vs. 23 +/- 1 mmHg, p = 0.02), an increase in mean bladder capacity (C = 1230 +/- 250 microl vs. 484 +/- 60 microl, p = 0.03), and a decrease in mean volume at first non-voiding contraction (67 +/- 16 microl vs. 110 +/- 24 microl, p = 0.02) compared to CTRL. Addition of SNS neither significantly affected P(det) (30 +/- 3 mm Hg vs. 35 +/- 2 mmHg, p = 0.2), nor C (630 +/- 90 microl vs. 1230 +/- 250 microl, p = 0.06) compared to BOO, but eliminated non-voiding contractions. Detrusor hypertrophy and fibrosis were both significantly greater in BOO vs. CTRL and vs. BOO/SNS. CONCLUSIONS: Partial BOO caused functional and structural changes in the rat bladder. SNS in obstructed rats prevents these alterations, without adversely affecting detrusor contractility.

Multiplex pathogen identification for polymicrobial urinary tract infections using biosensor technology: a prospective clinical study.

PURPOSE: Rapid diagnosis of urinary tract infection would have a significant beneficial impact on clinical management, particularly in patients with structural or functional urinary tract abnormalities who are highly susceptible to recurrent polymicrobial infections. We examined the analytical validity of an electrochemical biosensor array for rapid molecular diagnosis of urinary tract infection in a prospective clinical study in patients with neurogenic bladder. MATERIALS AND METHODS: The electrochemical biosensor array was functionalized with DNA probes against 16S rRNA of the most common uropathogens. Spinal cord injured patients at a Veterans Affairs hospital were recruited into the study. Urine samples were generally tested on the biosensor within 1 to 2 hours of collection. Biosensor results were compared with those obtained using standard clinical microbiology laboratory methods. RESULTS: We successfully developed a 1-hour biosensor assay for multiplex identification of pathogens. From July 2007 to December 2008 we recruited 116 patients, yielding a total of 109 urine samples suitable for analysis and comparison between biosensor assay and standard urine culture. Of the samples 74% were positive, of which 42% were polymicrobial. We identified 20 organisms, of which Escherichia coli, Pseudomonas aeruginosa and Enterococcus species were the most common. Biosensor assay specificity and positive predictive value were 100%. Pathogen detection sensitivity was 89%, yielding a 76% negative predictive value. CONCLUSIONS: To our knowledge we report the first prospective clinical study to successfully identify pathogens within a point of care time frame using an electrochemical biosensor platform. Additional efforts to improve the limit of detection and probe design are needed to further enhance assay sensitivity.

The effect of sildenafil citrate on bladder outlet obstruction: a mouse model.

OBJECTIVE: To investigate if sildenafil citrate can inhibit the functional and structural changes of the detrusor in a murine model of bladder outlet obstruction (BOO). Phosphodiesterase type 5 (PDE-5) inhibitors have recently been used for treating urinary symptoms associated with prostatic obstruction, but it is unclear whether PDE-5 inhibition acts on the prostatic urethra or the bladder. MATERIALS AND METHODS: In 18 male Balb/CAN mice, partial BOO was created and the mice allowed to survive for 6 weeks. Half of the mice (nine) were treated with oral sildenafil citrate daily (10 mg/kg) by oral lavage (BOO + V), and half (nine) were not (BOO). Six mice were used as sham-operated controls and received no sildenafil. The mice were assessed by urodynamics at baseline and after 6 weeks, with a measurement of volume at first uninhibited non-voiding contraction (V(DO1)), bladder capacity (BC), and detrusor pressure during void (Pdet). At 6 weeks, bladders were harvested, fixed and sectioned, and stained with haematoxylin and eosin (H&E) and trichrome stain. Detrusor muscle hypertrophy and fibrosis were evaluated on a scale of 1 (decreased) to 3 (increased), by two urologists and one pathologist unaware of the treatment group; the results were compared with those from normal controls. RESULTS: BOO mice had a significantly greater BC than control mice, with a mean (SD) of 153 (66) vs 58 (13) microL (P = 0.004). Treatment with sildenafil did not significantly alter BC. BOO caused an increase in Pdet compared to controls, with a mean (SD) of 25 (7) vs 12 (5) cm H2O. P(det) was not significantly different after treatment with sildenafil. The median V(DO1) as a percentage of BC was significantly lower in BOO than in control mice (20% vs 53%, P > 0.03) and increased significantly after sildenafil treatment (20% vs 44%, P = 0.04). BOO was associated with a greater bladder weight than in control mice, with a mean (SD) of 89 (32) vs 27 (6) mg (P = 0.001), which was decreased with sildenafil treatment, to 40 (14) vs 89 (32) mg (P = 0.013). BOO caused an increase in detrusor muscular hypertrophy vs control mice, with a median H&E score of 3 vs 2 (P = 0.01) and an increase in fibrosis vs control mice, with a median trichrome score of 3 vs 2 (P = 0.01). BOO + V mice had reduced muscular hypertrophy and fibrosis, with a median H&E score of 3 vs 2 (P = 0.01) and a median trichrome score of 3 vs 1 (P = 0.01). CONCLUSIONS: BOO mediates both functional and structural changes in the mouse bladder. Six weeks of obstruction caused an increase in BC, detrusor overactivity and voiding pressure, and mediated an increase in bladder weight, detrusor muscle hypertrophy and collagen deposition in the lamina propria and smooth muscle. Treatment with 6 weeks of oral sildenafil beginning at the time of BOO prevented the increase in detrusor overactivity without affecting voiding pressures, and prevented the increase in detrusor muscle hypertrophy and collagen deposition that otherwise occurred with BOO. It appears therefore that sildenafil citrate acts on the bladder rather than on the outlet.

Delivery of intercellular adhesion molecule-1 antisense oligonucleotides using a topical hydrogel tissue sealant in a murine partial nephrectomy/ischemia model.

OBJECTIVES: Ischemia/reperfusion injury is a leading cause of renal damage which can be improved with antisense oligonucleotide gene therapy. We have shown that polyethylene glycol (PEG) hydrogel, which also functions as a tissue sealant, is an effective topical delivery vehicle for oligonucleotides in a murine partial nephrectomy model. The objective of this study was to use and evaluate this method against intercellular adhesion molecule-1 (ICAM-1) to prevent tissue damage. METHODS: Sixty mice underwent left upper pole partial nephrectomy with 45 minutes of warm ischemia, randomized to treatment with 50 microg ICAM-1 antisense oligonucleotides embedded in PEG hydrogel, no therapy, or sham surgery. Kidneys were harvested at 24 hours and 3, 4, and 5 days. The specimens were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) for ICAM-1 messenger ribonucleic acid (mRNA), immunohistochemical staining for ICAM-1 protein, and standard histology. RESULTS: At 24 hours, qRT-PCR and immunohistochemistry data showed a significant reduction in ICAM-1 mRNA and protein expression in the antisense group versus the ischemia group, but no difference at 3 to 5 days. Histologically there was reduced inflammation and necrosis in the cortex at 24 hours. The outer and inner medulla also showed improvement at 3 to 5 days in the antisense group as opposed to the ischemia group. CONCLUSIONS: Topical PEG hydrogel delivery of antisense ICAM-1 oligonucleotides demonstrated decreased ICAM-1 mRNA expression, reduced ICAM-1 protein staining, and decreased cellular damage. The application of gene therapy through this novel topical delivery system holds potential for a highly specific, localized method of preventing tissue damage after ischemia/reperfusion injury.

Angiotensin II plays a role in acute murine experimental autoimmune cystitis.

OBJECTIVES: To investigate whether angiotensin II (AII) receptor antagonism decreases the inflammation and oedema in acute murine experimental autoimmune cystitis (EAC), as interstitial cystitis (IC) might have an autoimmune component and AII has been implicated in autoimmune-mediated vascular congestion, oedema and scarring. MATERIALS AND METHODS: Female Balb/cAN mice were divided into three treatment groups (eight in each group) that were autoimmunized with bladder homogenate to induce EAC. One group received an AII type 1 receptor (AT(1)) antagonist, one group an AII type 2 receptor (AT(2)) antagonist, and one group remained untreated (EAC). A control and sham-injected group were also included. After 10 weeks, bladders were removed, sectioned, and stained with haematoxylin and eosin. RESULTS: Grossly, there was no thickening or adhesions in the bladders of the control or sham-injected mice. In five of seven surviving EAC bladders, there were dense adhesions to surrounding peritoneal structures. There were also adhesions and bladder thickening in all of the AT(2) antagonist-treated mice (though in a milder form) but in only two of seven surviving AT(1) antagonist-treated mice. There was no inflammation or oedema in the sham and control groups. All the EAC bladders were inflamed, with submucosal oedema and urothelial detachment from the lamina propria. In the AT(1) antagonist-treated mice there was no inflammation or oedema. By contrast, all AT(2) antagonist-treated mice had moderate inflammation and minor detachment of the urothelium from the lamina propria. CONCLUSIONS: AT(1) receptor blockade ameliorated the inflammatory infiltration, submucosal oedema, and urothelial detachment associated with EAC in mice. This was achieved to a lesser extent by AT(2) receptor blockade. If some patients with IC have a pathophysiology similar to that of EAC mice, there might be potential benefit from AII receptor blockade.

The role of angiotensin II in stress urinary incontinence: A rat model.

AIMS: Pharmacological treatment for stress urinary incontinence (SUI) is limited to the use of non-selective alpha-agonists, which are often ineffective. Non-adrenergic mechanisms have also been implicated in urethral closure, including angiotensin II (Ang-II), which has been demonstrated throughout the urinary tract. We investigate the role of Ang-II in urethral tone in a rat model of SUI. METHODS: Abdominal leak point pressure (ALPP) and retrograde urethral pressure profilometry (RLPP) were measured in 70 female virgin rats. Thirty rats underwent pudendal nerve injury (PNT), 30 had circumferential urethrolysis (U-Lys), and 10 had sham surgery. Rats received daily doses of Angiotensin Type 1 (AT-1) receptor inhibitor (20 mg/kg), Angiotensin Type 2 (AT-2) receptor antagonist (10 mg/kg), or Ang-II (2 mg/kg). RESULTS: Following U-Lys, RLPP and ALPP decreased from 21.4 +/- 2.0 and 39.2 +/- 3.3 mm Hg, to 13.1 +/- 1.5 and 21.6 +/- 1.9 mmHg, respectively (P < 0.01). After PNT, RLPP, and ALPP decreased from 21.0 +/- 1.6 and 41.9 +/- 3.0 mmHg to 13.1 +/- 1.5 and 24.7 +/- 3.3 mmHg, respectively (P < 0.01). AT-1 inhibitor caused significant decrease in RLPP and ALPP from 21.0 +/- 6.2 and 41.8 +/- 9.4 mmHg, to 12.0 +/- 3.8 and 25.6 +/- 6.6 mmHg, respectively (P < 0.01). Likewise, AT-2 treatment reduced RLPP and ALPP from 21.4 +/- 6.3 and 40.1 +/- 1.7 mmHg, to 13.5 +/- 5.7 and 31.0 +/- 7.2 mmHg, respectively (P < 0.01). Following surgery, Ang-II administration restored RLPP and ALPP to baseline presurgical values. CONCLUSIONS: AT-1 and AT-2 receptor inhibition significantly lowers urethral resistance, comparable to either neurogenic or urethrolytic injury. Ang-II treatment restored urethral tone in rats with intrinsic sphincter dysfunction. Ang II appears to serve a functional role in the maintenance of urethral tone and stress continence.

Novel delivery of oligonucleotides using a topical hydrogel tissue sealant in a murine partial nephrectomy model.

PURPOSE: Ischemia/reperfusion injury is a leading cause of renal damage and antisense gene therapy has been shown to ameliorate its effects. However, this approach has been limited by current delivery methods that require high concentrations of intravenous nucleic acids lacking specificity for targeting tissues. To overcome these limitations we developed a novel murine partial nephrectomy model to evaluate polyethylene-glycol (PEG) hydrogel tissue sealant as a topical oligonucleotide delivery system. MATERIALS AND METHODS: A total of 18 male C57BL/6 mice underwent left partial nephrectomy with vascular occlusion. Hydrogel primer and then sealant were applied to the cut surface and photopolymerized. Using this method 16 additional mice received hydrogel primer mixed with Cy5 labeled fluorescent oligonucleotide (10 to 100 microg). Kidneys were harvested at various time points and assessed for oligonucleotide penetration using fluorescence microscopy. RESULTS: A survival rate of 100% (34 subjects) was obtained using this mouse model of partial nephrectomy. PEG hydrogel provided adequate protection against renal hematoma and intraperitoneal blood. Fluorescent images revealed that 50 microg was the minimum dose resulting in complete progressive cellular penetration with time. In addition to direct diffusion from the application site, movement of oligonucleotide through the subcapsular space into the cortex was an observed mechanism of distribution. CONCLUSIONS: A murine partial nephrectomy model was successfully created using PEG hydrogel. In addition to achieving hemostasis, hydrogel served as a successful depot for delivering oligonucleotides throughout the kidney.