Applications of EUS for the Molecular Characterization and Treatment of Gastrointestinal Diseases – A Review of the Recent Medical Literature

Introduction

With the development of linear echoendoscopes, interventional endoscopic ultrasonography (EUS) has become a clinical reality. The widest application for interventional EUS is EUS-guided fine-needle aspiration (FNA). EUS-guided FNA is performed for sampling pancreatic masses, submucosal tumors, lymph nodes, perigastrointestinal masses, adrenal glands, and hilar lesions.[1–10] However, there has been ongoing interest by endosonographers in applying EUS-guided FNA beyond the already established applications.

Molecular Studies on EUS-Guided FNA

Cytopathologic analysis of EUS-guided FNA material has been the conventional method for making a diagnosis in lesions targeted by EUS. However, recently there has been ongoing interest in molecular and/or genetic analysis of material that is aspirated during EUS-guided FNA in an attempt to go beyond conventional cytologic evaluation. One application of this approach is to perform polymerase chain reaction (PCR) analysis on aspirated material collected during EUS-guided puncture of lymph nodes in order to detect micrometastases. In a study of 34 lymph nodes that were subjected to EUS-FNA, real-time PCR using a panel of 5 lung cancer-associated genes was performed.[11] Of 8 pathologically positive lymph nodes, 4 candidate markers were overexpressed. Twenty-six lymph nodes were pathologically negative; 46% (n = 12) overexpressed at least 1 marker, whereas 6 of the 26 (23%) overexpressed at least 2 markers. Thus, EUS-guided FNA with real-time PCR of 5 lung cancer-associated genes showed 50% positivity for micrometastases in lymph nodes that would be considered pathologically nonmalignant by conventional criteria. In a follow-up study[12] of the same group of 87 patients with non-small-cell lung cancer (NSCLC), mediastinal lymph nodes were sampled by EUS-FNA and evaluated by standard cytopathology and real-time reverse transcriptase (RT)-PCR. Messenger RNA was extracted and real-time RT-PCR was used to quantitate the expression of 6 lung cancer-associated genes (ie, CEA [carcinoembryonic antigen], CK [cytokeratin]19, KS 1/4, lunx, muc 1, and PDEF). Among the cytology-positive lymph nodes (n = 27), the expression of the KS1/4 gene was greater than its respective clinical threshold in 25 of 27 samples (93%), making this the most sensitive marker for the detection of metastatic NSCLC. At least 1 of the 6 lung cancer-associated genes was overexpressed in 18 of 61 cytology-negative patients (30%), of which KS1/4 was overexpressed in 15 of 61 patients (25%). The study authors predicted that some of the patients in the cytology-negative/marker-positive group will have high rates of NSCLC recurrence. Widespread availability of these new techniques coupled with prospective, randomized, multicenter outcomes trials is required to determine the impact of molecular marker positivity within mediastinal lymph nodes upon treatment modalities and survival.[13]

Pellise and colleagues[14] reported their efforts to detect micrometastases beyond conventional cytologic analysis. These investigators attempted to evaluate the feasibility and utility of hypermethylation gene promoter analysis for detecting micrometastases in lymph nodes. Material collected by EUS-guided FNA from lymph nodes of patients with lung and gastrointestinal cancer was subjected to methylation analyses of the MGMT, P161Nk4a, and P14ARF gene promoters of CpG islands (unmethylated regions of the genome that are associated with the 5' ends of many genes) using methylation-specific PCR. The combination of conventional cytology with methylation-specific PCR increased the sensitivity but reduced the specificity for detection of lymph node micrometastases.

Molecular marker studies have also been initiated in the setting of pancreatic disease. Anderson and colleagues[15] reported the expression level of specific pancreatic cancer genes (SPC1, SPC2, and SPC3) using quantitative PCR in samples obtained during EUS-guided FNA of pancreas. All 3 nonmarker pancreatic cancer genes were elevated in 6 pancreatic adenocarcinoma specimens and 2 cystic pancreatic neoplasms. Samples from resected pancreatic specimens with chronic pancreatitis failed to show expression of the 3 biomarker genes. Khalid and colleagues[16] have recently investigated the utility of free-floating DNA in cyst fluid to help in the preoperative characterization and differentiation of cystic tumors of the pancreas. These study authors subjected pancreatic cyst fluid obtained from EUS-guided FNA to PCR amplification; a panel of 15 microsatellite allelic loss markers situated at 1p, 3p, 5q, 9p, 10q, and 17q were studied and K-ras-2 point mutation analysis was performed. They found significantly more allelic losses at critical sites in malignant cysts compared with in benign cysts. Raimondo and colleagues[17] studied the utility of combining structural information obtained by EUS with secretin-stimulated pancreatic juice analysis for interleukin-8 and intercellular adhesion molecule-1, with the goal of improving the clinician's ability to diagnose chronic pancreatitis.

Molecular studies on EUS-FNA material are promising investigational techniques that are not yet ready for routine clinical practice, but in the future may provide diagnostic and prognostic information that is beyond routine conventional cytology.

EUS-Guided Ablation

Many groups are investigating the potential utility of using EUS to deliver various forms of ablative energy to target tumors. There has been ongoing interest in using high-intensity focused ultrasound (HIFU) for tumor ablation; this technology involves focusing ultrasound waves in a narrow focal zone to develop high-intensity ultrasound fields.[18] Using animal models, Prat and colleagues[19] demonstrated that HIFU could be applied successfully with small transducers adaptable for use during endoscopy. This same group has also reported pilot results of a human trial using HIFU during endoscopic retrograde cholangiopancreatography (ERCP).[20] In this study, 10 patients with cholangiocarcinoma or ampullary carcinoma were treated by application of HIFU. Subsequent surgery in 2 patients revealed extensive coagulation necrosis in 1 and no malignancy in the other. One patient had complete regression of cholangiocarcinoma, whereas partial response was noted in 4 other patients.

A group from Boston investigated the feasibility of performing radiofrequency ablation in the pancreas under EUS guidance in a swine model.[21] A 19-gauge needle was inserted into the pancreas via the transgastric route under real-time EUS guidance. Necropsy revealed discrete 8- to 12-mm spherical foci of coagulation necrosis. The study authors suggested potential clinical applications of EUS-guided radiofrequency ablation for management of small functioning neuroendocrine tumors and for palliation of unresectable pancreatic adenocarcinoma. This same group also reported an attempt at EUS-guided photodynamic therapy in a swine model.[22] The swine were injected intravenously with porfimer sodium. EUS-guided puncture of various organs was performed with a 19-gauge needle; a miniature quartz optical fiber was placed through the needle into the tissue, and photodynamic therapy was performed. The mean areas of necrosis induced by photodynamic therapy in various organs were 3.6 mm² in pancreas, 3.3 mm² in liver, 3.2 mm² in kidneys, and 8.5 mm² in spleen.

Investigators from Germany[23,24] studied the use of an EUS-based radiation target simulation method for allowing application of afterloading needles with precise control and to optimize radiation target geometry in patients with anal cancer. The study authors investigated this method of radiation treatment for anal cancer. The study authors reported 42 EUS-guided afterloading procedures in 18 patients with complete remission achieved in all cases. Median follow-up of 24 months revealed 2 anal cancer recurrences. More recent results reported by this group for 36 patients again resulted in complete initial remission in all cases. Five recurrent tumors (13.9%) were detected in a mean follow-up of 44 months. The investigators concluded that this method allowed them to achieve a high rate of cure, low rate of complications, and minimized the chances of recurrence.[23,24]

EUS-guided ablation technology is a promising technique that is generally still investigational but which may be used in the future alone or in combination with other treatments for cancer therapy.

EUS-Guided Fine-Needle Injection

There is ongoing interest in using the precise targeting abilities of EUS to deliver or inject a therapeutic agent to a specific anatomic location under EUS guidance. This technology has been termed EUS-guided fine-needle injection (FNI). EUS-guided celiac ganglion neurolysis or block is performed under EUS guidance,[25] and botulinum toxin may be precisely injected into the lower esophageal sphincter under EUS control.[26] A number of studies involving the application of this technology to inject therapeutic agents under EUS guidance are currently under way.

Chang and colleagues[27] conducted a clinical trial of local immunotherapy by injecting activated T-lymphocytes into unresectable pancreatic cancers by EUS-guided FNI. The median survival in this study was 13.2 months. Another pilot study[28] investigated the tolerability and efficacy of EUS-guided FNI of a modified adenovirus into pancreatic adenocarcinomas. In this trial, patients underwent 8 sessions of ONYX-015 virus injection over 8 weeks with concomitant gemcitabine. Diluted virus was injected in 1–2 mL aliquots into the pancreatic tumor in a fan-like fashion with a fine needle under real-time EUS guidance using a linear-array echoendoscope. After combination therapy, 2 patients had partial regressions of the injected tumor, 2 had minor responses, 6 had stable disease, and 11 had progressive disease or had to go off study because of treatment toxicity.

Chang and colleagues[29] have also recently reported another attempt at EUS-guided FNI for pancreatic cancer. The study authors injected TNFerade into pancreatic cancer using EUS-guided (n = 17) or percutaneous injection (n = 20). TNFerade is a replication-deficient adenovector containing the human tumor necrosis factor-alpha gene that is regulated by the radiation-inducible promoter Egr-1. Some signs of local control of disease and progression-free survival were seen during a short-term follow-up of 3 months.

Other attempts at applying EUS-guided FNI technology includ EUS-guided fine-needle tattooing for better intraoperative localization of neuroendocrine tumors[30]; EUS-guided superior hypogastric plexus neurolysis[31]; and EUS-guided transgastric injection of N-2-butyl-cyanoacrylate glue into pancreatic duct to control pancreatic ascites.[32] Another group has recently reported control of traumatic chylothorax with EUS-guided thoracic duct injection sclerotherapy.[33]

There is emerging interest in the use of EUS-guided injection of alcohol as antitumor therapy. Two isolated reports have used EUS-guided alcohol injection to ablate a solitary hepatic metastasis[34] and a submucosal gastric stromal tumor.[35] Even more interesting is an attempt aimed at treating cystic tumors of the pancreas using EUS-guided ethanol lavage. Gan and colleagues[36] performed EUS-guided ethanol lavage with 5% to 80% ethanol for 3–5 minutes in 24 patients with cystic lesions of the pancreas. Three patients underwent subsequent surgery, with surgical specimens showing denuded epithelium and no evidence of pancreatitis. Follow-up imaging in 8 other patients revealed resolution of the lesion in 5 and persistence of the lesion in 3.

EUS-guided antitumor therapy is in its infancy, but has a number of potential applications in the future as it holds promise as a targeting device for specific local injection of a therapeutic or biologic agent. The question is not whether EUS can deliver a specific agent to a targeted location into the pancreas or elsewhere; what we need to know is the ideal or optimal agent to be delivered via EUS into a tumor.

EUS-Guided Anastomoses

Due to the ability of EUS to image beyond the gut lumen as well as the capability to perform intervention under EUS-guidance, there has been ongoing interest in evaluating the utility of EUS in creating various forms of anastomoses in the gastrointestinal tract. There has been continued interest in the development of techniques for real-time EUS-guided puncture of a pseudocyst. Given the ongoing efforts by a few groups, along with availability of larger-channel echoendoscopes, EUS-guided pseudocyst drainage with placement of a prosthesis for drainage through an echoendoscope is now possible.[37–39] A series of 35 patients with pancreatic pseudocyst or abscess revealed an 89% success rate for drainage performed exclusively using an echoendoscope.[40] Choledochoduodenostomy may be performed with EUS, as reported in a patient with failed ERCP, in whom placement of a transduodenal stent into the dilated/obstructed biliary system was performed under EUS guidance.[41] EUS-guided pancreaticogastrostomy was reported in 4 patients with chronic pancreatitis and obstructed/dilated pancreatic duct.[42] Hepaticogastrostomy has been attempted under EUS guidance in a swine model.[43] Endoscopic suturing devices provide another opportunity for combination of technology with the capabilities of EUS.

Fritscher-Ravens and colleagues[44] recently reported a through-the-scope device for endoscopic suturing and tissue approximation under EUS control. Two other groups recently reported transrectal EUS-guided drainage of pelvic and diverticular abscesses.[45,46] Transesophageal mediastinal abscess drainage was reported by another group.[47] EUS-guided access to the vascular and lymphatic system has also been attempted recently in animal models.[48,49]

Some of the aforementioned techniques have already been performed in patients (pseudocyst drainage, choledochoduodenostomy, pancreaticogastrostomy, etc.), whereas others have only been attempted in animal models with hopefully clinical application in patients in the future.

Concluding Remarks

There appear to be varied and robust attempts at using interventional endosonography for a variety of therapeutic applications. The minimally invasive interventional options using EUS may serve as an alternative to many indications traditionally treated with conventional surgery. The ability to study molecular markers in material obtained by EUS-FNA and the precise targeting capabilities of EUS hold promise for this technology to play an important role in translational research as recent advances in proteomics, genomics, and gene therapy at the bench are studied for clinical application in patients.