Maliha Zahid

Pancreatic cyst fluid DNA analysis in evaluating pancreatic cysts: a report of the PANDA study

BACKGROUND The role of pancreatic cyst fluid DNA analysis in evaluating pancreatic cysts remains unclear. OBJECTIVE Our purpose was to evaluate the utility of a detailed DNA analysis of pancreatic cyst fluid to diagnose mucinous and malignant cysts. DESIGN Prospective, multicenter study. PATIENTS Patients with pancreatic cysts presenting for EUS evaluation. INTERVENTION EUS-guided pancreatic cyst aspirates cytology evaluation, carcinoembryonic antigen (CEA) level determination, and a detailed DNA analysis; incorporating DNA quantification, k-ras mutation and multiple allelic loss analysis, mutational amplitude, and sequence determination. MAIN OUTCOME MEASUREMENTS Cyst fluid analysis compared with surgical pathologic or malignant cytologic examination. RESULTS The study cohort consisted of 113 patients with 40 malignant, 48 premalignant, and 25 benign cysts. Cyst fluid k-ras mutation was helpful in the diagnosis of mucinous cysts (odds ratio 20.9, specificity 96%), whereas receiver-operator characteristic curve analysis indicated optimal cutoff points for allelic loss amplitude (area under the curve [AUC] 0.79; optimal value > 65%) and CEA (AUC 0.74; optimal value >148 ng/mL). Components of DNA analysis detecting malignant cysts included allelic loss amplitude over 82% (AUC 0.9) and high DNA amount (optical density ratio >10, AUC 0.79). The criteria of a high amplitude k-ras mutation followed by allelic loss showed maximum specificity (96%) for malignancy. All malignant cysts with negative cytologic evaluation (10/40) could be diagnosed as malignant by using DNA analysis. LIMITATIONS Limited follow-up, selection bias. CONCLUSIONS Elevated amounts of pancreatic cyst fluid DNA, high-amplitude mutations, and specific mutation acquisition sequences are indicators of malignancy. The presence of a k-ras mutation is also indicative of a mucinous cyst. DNA analysis should be considered when cyst cytologic examination is negative for malignancy.

The Role of Pancreatic Cyst Fluid Molecular Analysis in Predicting Cyst Pathology

BACKGROUND & AIMS Current methods to detect malignancy in mucinous cystic neoplasms of the pancreas remain inadequate. The role of detailed molecular analysis in this context was investigated. METHODS Endoscopic ultrasound-guided pancreatic cyst aspirates were prospectively collected during a period of 19 months and studied for cytology, carcinoembryonic antigen level, and molecular analysis. Molecular evaluation incorporated DNA quantification (amount and quality), k-ras point mutation, and broad panel tumor suppressor linked microsatellite marker allelic loss analysis by using fluorescent capillary electrophoresis. The sequence of mutation acquisition was also calculated on the basis of a clonal expansion model, and comparison was made to the final pathology. RESULTS Thirty-six cysts with confirmed histology were analyzed. There were 11 malignant, 15 premalignant, and 10 benign cysts. Malignant cysts could be differentiated from premalignant cysts on the basis of fluid carcinoembryonic antigen level (P=.034), DNA quality (P=.009), number of mutations (P=.002), and on the sequence of mutations acquired (P<.001). Early k-ras mutation followed by allelic loss was the most predictive of a malignant cyst (sensitivity, 91%; specificity, 93%). CONCLUSIONS Malignant cyst fluid contains adequate DNA to allow mutational analysis. A first hit k-ras mutation followed by allelic loss is most predictive of the presence of malignancy in a pancreatic cyst. This approach should serve as an ancillary tool to the conventional work-up of pancreatic cysts. Cumulative amount and timing of detectable mutational damage can assist in diagnosis and clinical management.

Endoscopic Ultrasound Fine Needle Aspirate DNA Analysis to Differentiate Malignant and Benign Pancreatic Masses

OBJECTIVES:Accurate diagnosis of malignant and benign pancreatic masses can be challenging, potentially delaying treatment for cancer and subjecting patients with benign disease to unnecessary surgery. Endoscopic ultrasound fine needle aspirate (EUS-FNA) of pancreatic masses remains inconclusive in a subset of patients. The role of EUS-FNA molecular analysis in this context is studied.METHODS:Patients with benign pancreatic masses (6 cases, 4 autoimmune pancreatitis, 2 focal chronic pancreatitis) and malignant pancreatic masses (15) with inconclusive cytology (5 cases) and positive cytology (10 controls) were selected. All cases had definitive pathology. Representative cells were microdissected from each EUS-FNA sample and subjected to PCR for analysis of 16 microsatellite allele loss markers situated at 1p, 3p, 5q, 9p, 9q, 10q, 17p, 17q, 21q, and 22q. Loss of heterozygosity analysis used fluorescent capillary electrophoresis for quantitative determination of allelic imbalance. k-ras-2 point mutation analysis was also performed. Mean fractional mutation rate (FMR) was calculated and compared for each group.RESULTS:All malignant cases carried multiple mutations (FMR 0.50), regardless of positive cytology (FMR 0.52) or suspicious cytology (FMR 0.47) (p = NS). Five of the 6 benign cases carried no mutations whereas 1 case of autoimmune pancreatitis and coexisting PanIN lesions exhibited a k-ras mutation (FMR 0.01). The mean FMR for the malignant and benign samples was significantly different (p < 0.0001).CONCLUSIONS:Broad panel microsatellite loss and k-ras point mutation analysis can be reliably performed on EUS-FNA samples from pancreatic masses and improves the diagnostic accuracy. Furthermore, it accurately differentiates between malignant and benign pancreatic masses.

A potential role of distinctively delayed blood clearance of recombinant adeno-associated virus serotype 9 in robust cardiac transduction.

Recombinant adeno-associated virus serotype 9 (rAAV9) vectors show robust in vivo transduction by a systemic approach. It has been proposed that rAAV9 has enhanced ability to cross the vascular endothelial barriers. However, the scientific basis of systemic administration of rAAV9 and its transduction mechanisms have not been fully established. Here, we show indirect evidence suggesting that capillary walls still remain as a significant barrier to rAAV9 in cardiac transduction but not so in hepatic transduction in mice, and the distinctively delayed blood clearance of rAAV9 plays an important role in overcoming this barrier, contributing to robust cardiac transduction. We find that transvascular transport of rAAV9 in the heart is a capacity-limited slow process and occurs in the absence of caveolin-1, the major component of caveolae that mediate endothelial transcytosis. In addition, a reverse genetic study identifies the outer region of the icosahedral threefold capsid protrusions as a potential culprit for rAAV9s delayed blood clearance. These results support a model in which the delayed blood clearance of rAAV9 sustains the capacity-limited slow transvascular vector transport and plays a role in mediating robust cardiac transduction, and provide important implications in AAV capsid engineering to create new rAAV variants with more desirable properties.

Identification of a Cardiac Specific Protein Transduction Domain by In Vivo Biopanning Using a M13 Phage Peptide Display Library in Mice

Background A peptide able to transduce cardiac tissue specifically, delivering cargoes to the heart, would be of significant therapeutic potential for delivery of small molecules, proteins and nucleic acids. In order to identify peptide(s) able to transduce heart tissue, biopanning was performed in cell culture and in vivo with a M13 phage peptide display library. Methods and Results A cardiomyoblast cell line, H9C2, was incubated with a M13 phage 12 amino acid peptide display library. Internalized phage was recovered, amplified and then subjected to a total of three rounds of in vivo biopanning where infectious phage was isolated from cardiac tissue following intravenous injection. After the third round, 60% of sequenced plaques carried the peptide sequence APWHLSSQYSRT, termed cardiac targeting peptide (CTP). We demonstrate that CTP was able to transduce cardiomyocytes functionally in culture in a concentration and cell-type dependent manner. Mice injected with CTP showed significant transduction of heart tissue with minimal uptake by lung and kidney capillaries, and no uptake in liver, skeletal muscle, spleen or brain. The level of heart transduction by CTP also was greater than with a cationic transduction domain. Conclusions Biopanning using a peptide phage display library identified a peptide able to transduce heart tissue in vivo efficiently and specifically. CTP could be used to deliver therapeutic peptides, proteins and nucleic acid specifically to the heart.

Airway ciliary dysfunction and sinopulmonary symptoms in patients with congenital heart disease

RATIONALE Patients with congenital heart disease with heterotaxy exhibit a high prevalence of abnormal airway ciliary motion and low nasal nitric oxide, characteristics associated with primary ciliary dyskinesia, a reflection of the role of motile cilia in airway clearance and left-right patterning. OBJECTIVES To assess the potential broader clinical significance of airway ciliary dysfunction in congenital heart disease, we assessed the prevalence of ciliary dysfunction versus respiratory symptoms in patients with congenital heart disease with or without heterotaxy. METHODS Patients with a broad spectrum of congenital heart disease were recruited (n = 218), 39 with heterotaxy. Nasal nitric oxide measurements and nasal biopsies for ciliary motion video microscopy were conducted. Sinopulmonary symptoms were reviewed by questionnaire. MEASUREMENTS AND MAIN RESULTS A high prevalence of ciliary motion defects (51.8%) and low or borderline low nasal nitric oxide levels (35.5%) were observed in patients with congenital heart disease with or without heterotaxy. Patients with ciliary motion defects or low nasal nitric oxide showed increased sinopulmonary symptoms, with most respiratory symptoms seen in those with both abnormal ciliary motion and low nitric oxide. Multivariate analysis showed that abnormal ciliary motion and low nasal nitric oxide were more important in determining risk of sinopulmonary symptoms than heterotaxy status. CONCLUSIONS Patients with congenital heart disease without heterotaxy exhibit a high prevalence of abnormal ciliary motion and low nasal nitric oxide. This was associated with more sinopulmonary symptoms. These findings suggest that patients with a broad spectrum of congenital heart disease and respiratory symptoms may benefit from screening for ciliary dysfunction and implementation of medical interventions to reduce sinopulmonary morbidities.

Cell-type specific penetrating peptides: therapeutic promises and challenges.

Cell penetrating peptides (CPP), also known as protein transduction domains (PTD), are small peptides able to carry peptides, proteins, nucleic acid, and nanoparticles, including viral particles, across the cellular membranes into cells, resulting in internalization of the intact cargo. In general, CPPs can be broadly classified into tissue-specific and non-tissue specific peptides, with the latter further sub-divided into three types: (1) cationic peptides of 6–12 amino acids in length comprised predominantly of arginine, lysine and/or ornithine residues; (2) hydrophobic peptides such as leader sequences of secreted growth factors or cytokines; and (3) amphipathic peptides obtained by linking hydrophobic peptides to nuclear localizing signals. Tissue-specific peptides are usually identified by screening of large peptide phage display libraries. These transduction peptides have the potential for a myriad of diagnostic as well as therapeutic applications, ranging from delivery of fluorescent or radioactive compounds for imaging, to delivery of peptides and proteins of therapeutic potential, and improving uptake of DNA, RNA, siRNA and even viral particles. Here we review the potential applications as well as hurdles to the tremendous potential of these CPPs, in particular the cell-type specific peptides.

DNAH6 and Its Interactions with PCD Genes in Heterotaxy and Primary Ciliary Dyskinesia.

Heterotaxy, a birth defect involving left-right patterning defects, and primary ciliary dyskinesia (PCD), a sinopulmonary disease with dyskinetic/immotile cilia in the airway are seemingly disparate diseases. However, they have an overlapping genetic etiology involving mutations in cilia genes, a reflection of the common requirement for motile cilia in left-right patterning and airway clearance. While PCD is a monogenic recessive disorder, heterotaxy has a more complex, largely non-monogenic etiology. In this study, we show mutations in the novel dynein gene DNAH6 can cause heterotaxy and ciliary dysfunction similar to PCD. We provide the first evidence that trans-heterozygous interactions between DNAH6 and other PCD genes potentially can cause heterotaxy. DNAH6 was initially identified as a candidate heterotaxy/PCD gene by filtering exome-sequencing data from 25 heterotaxy patients stratified by whether they have airway motile cilia defects. dnah6 morpholino knockdown in zebrafish disrupted motile cilia in Kupffer’s vesicle required for left-right patterning and caused heterotaxy with abnormal cardiac/gut looping. Similarly DNAH6 shRNA knockdown disrupted motile cilia in human and mouse respiratory epithelia. Notably a heterotaxy patient harboring heterozygous DNAH6 mutation was identified to also carry a rare heterozygous PCD-causing DNAI1 mutation, suggesting a DNAH6/DNAI1 trans-heterozygous interaction. Furthermore, sequencing of 149 additional heterotaxy patients showed 5 of 6 patients with heterozygous DNAH6 mutations also had heterozygous mutations in DNAH5 or other PCD genes. We functionally assayed for DNAH6/DNAH5 and DNAH6/DNAI1 trans-heterozygous interactions using subthreshold double-morpholino knockdown in zebrafish and showed this caused heterotaxy. Similarly, subthreshold siRNA knockdown of Dnah6 in heterozygous Dnah5 or Dnai1 mutant mouse respiratory epithelia disrupted motile cilia function. Together, these findings support an oligogenic disease model with broad relevance for further interrogating the genetic etiology of human ciliopathies.

The complex genetics of hypoplastic left heart syndrome

Congenital heart disease (CHD) affects up to 1% of live births. Although a genetic etiology is indicated by an increased recurrence risk, sporadic occurrence suggests that CHD genetics is complex. Here, we show that hypoplastic left heart syndrome (HLHS), a severe CHD, is multigenic and genetically heterogeneous. Using mouse forward genetics, we report what is, to our knowledge, the first isolation of HLHS mutant mice and identification of genes causing HLHS. Mutations from seven HLHS mouse lines showed multigenic enrichment in ten human chromosome regions linked to HLHS. Mutations in Sap130 and Pcdha9, genes not previously associated with CHD, were validated by CRISPR–Cas9 genome editing in mice as being digenic causes of HLHS. We also identified one subject with HLHS with SAP130 and PCDHA13 mutations. Mouse and zebrafish modeling showed that Sap130 mediates left ventricular hypoplasia, whereas Pcdha9 increases penetrance of aortic valve abnormalities, both signature HLHS defects. These findings show that HLHS can arise genetically in a combinatorial fashion, thus providing a new paradigm for the complex genetics of CHD.

Respiratory motile cilia dysfunction in a patient with cranioectodermal dysplasia

Ciliopathies such as cranioectodermal dysplasia, Sensenbrenner syndrome, short‐rib polydactyly, and Jeune syndrome are associated with respiratory complications arising from rib cage dysplasia. While such ciliopathies have been demonstrated to involve primary cilia defects, we show motile cilia dysfunction in the airway of a patient diagnosed with cranioectodermal dysplasia. While this patient had mild thoracic dystrophy not requiring surgical treatment, there was nevertheless newborn respiratory distress, restrictive airway disease with possible obstructive airway involvement, repeated respiratory infections, and atelectasis. High‐resolution videomicroscopy of nasal epithelial biopsy showed immotile/dyskinetic cilia and nasal nitric oxide was reduced, both of which are characteristics of primary ciliary dyskinesia, a sinopulmonary disease associated with mucociliary clearance defects due to motile cilia dysfunction in the airway. Exome sequencing analysis of this patient identified compound heterozygous mutations in WDR35, but no mutations in any of the 30 known primary ciliary dyskinesia genes or other cilia‐related genes. Given that WDR35 is only known to be required for primary cilia function, we carried out WDR35 siRNA knockdown in human respiratory epithelia to assess the role of WDR35 in motile cilia function. This showed WDR35 deficiency disrupted ciliogenesis in the airway, indicating WDR35 is also required for formation of motile cilia. Together, these findings suggest patients with WDR35 mutations have an airway mucociliary clearance defect masked by their restrictive airway disease.