Josephine Mitcham

Characterization of CD4+CD25+ Regulatory T Cells in Patients Treated With High-Dose Interleukin-2 for Metastatic Melanoma or Renal Cell Carcinoma

PURPOSE To characterize the number and functional status of CD4+CD25+ regulatory T cells (Tregs) in patients with metastatic melanoma (MM) and renal cell carcinoma (RCC) treated with high-dose bolus interleukin-2 (IL-2). PATIENTS AND METHODS Patients with MM or RCC treated with high-dose bolus IL-2 (600,000 IU/kg every 8 hours) at a single center provided pre- and post-treatment whole blood specimens. Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, separated into cellular subsets, and analyzed by flow cytometry or used for in vitro proliferation assays. RESULTS Between September 2003 and July 2005 57 patients were enrolled in the study with 48 patients available for analysis (45 MM, 12 RCC). Tregs were defined as CD4+CD25(hi) T cells, and this subset was significantly elevated in the cancer patients compared with normal donors (7.75% v 2.24%). The CD4(+)CD25(hi) T-cell pool in the patients constitutively expressed intracellular FoxP3, CTLA-4, and produced high amounts of IL-10. The Tregs were CCR7+ with 50% representing naïve and 50% central-memory T cells. The cells were functionally suppressive in mixed in vitro proliferation assays. Following IL-2 administration, the number and frequency of Tregs increased in patients with progressive disease but returned to normal levels in patients with objective clinical responses. CONCLUSION The number of Tregs, defined as CD4+CD25(hi) T cells is increased in patients with MM and RCC. High-dose IL-2 resulted in a significant decrease of Tregs in those patients achieving an objective clinical response to IL-2 therapy.

Targeting the local tumor microenvironment with vaccinia virus expressing B7.1 for the treatment of melanoma

Immunotherapy for the treatment of metastatic melanoma remains a major clinical challenge. The melanoma microenvironment may lead to local T cell tolerance in part through downregulation of costimulatory molecules, such as B7.1 (CD80). We report the results from the first clinical trial, to our knowledge, using a recombinant vaccinia virus expressing B7.1 (rV-B7.1) for monthly intralesional vaccination of accessible melanoma lesions. A standard 2-dose-escalation phase I clinical trial was conducted with 12 patients. The approach was well tolerated with only low-grade fever, myalgias, and fatigue reported and 2 patients experiencing vitiligo. An objective partial response was observed in 1 patient and disease stabilization in 2 patients, 1 of whom is alive without disease 59 months following vaccination. All patients demonstrated an increase in postvaccination antibody and T cell responses against vaccinia virus. Systemic immunity was tested in HLA-A*0201 patients who demonstrated an increased frequency of gp100 and T cells specific to melanoma antigen recognized by T cells 1 (MART-1), also known as Melan-A, by ELISPOT assay following local rV-B7.1 vaccination. Local immunity was evaluated by quantitative real-time RT-PCR, which suggested that tumor regression was associated with increased expression of CD8 and IFN-gamma. The local delivery of vaccinia virus expressing B7.1 was well tolerated and represents an innovative strategy for altering the local tumor microenvironment in patients with melanoma.

In vivo diagnostic accuracy of high resolution microendoscopy in differentiating neoplastic from non-neoplastic colorectal polyps: a prospective study

OBJECTIVES:High-resolution microendoscopy (HRME) is a low-cost, “optical biopsy” technology that allows for subcellular imaging. The purpose of this study was to determine the in vivo diagnostic accuracy of the HRME for the differentiation of neoplastic from non-neoplastic colorectal polyps and compare it to that of high-definition white-light endoscopy (WLE) with histopathology as the gold standard.METHODS:Three endoscopists prospectively detected a total of 171 polyps from 94 patients that were then imaged by HRME and classified in real-time as neoplastic (adenomatous, cancer) or non-neoplastic (normal, hyperplastic, inflammatory).RESULTS:HRME had a significantly higher accuracy (94%), specificity (95%), and positive predictive value (PPV, 87%) for the determination of neoplastic colorectal polyps compared with WLE (65%, 39%, and 55%, respectively). When looking at small colorectal polyps (less than 10 mm), HRME continued to significantly outperform WLE in terms of accuracy (95% vs. 64%), specificity (98% vs. 40%) and PPV (92% vs. 55%). These trends continued when evaluating diminutive polyps (less than 5 mm) as HRMEs accuracy (95%), specificity (98%), and PPV (93%) were all significantly greater than their WLE counterparts (62%, 41%, and 53%, respectively).CONCLUSIONS:In conclusion, this in vivo study demonstrates that HRME can be a very effective modality in the differentiation of neoplastic and non-neoplastic colorectal polyps. A combination of standard white-light colonoscopy for polyp detection and HRME for polyp classification has the potential to truly allow the endoscopist to selectively determine which lesions can be left in situ, which lesions can simply be discarded, and which lesions need formal histopathologic analysis.

Low-Cost High-Resolution Microendoscopy for the Detection of Esophageal Squamous Cell Neoplasia: An International Trial

BACKGROUND & AIMS Esophageal squamous cell neoplasia has a high mortality rate as a result of late detection. In high-risk regions such as China, screening is performed by Lugols chromoendoscopy (LCE). LCE has low specificity, resulting in unnecessary tissue biopsy with a subsequent increase in procedure cost and risk. The purpose of this study was to evaluate the accuracy of a novel, low-cost, high-resolution microendoscope (HRME) as an adjunct to LCE. METHODS In this prospective trial, 147 consecutive high-risk patients were enrolled from 2 US and 2 Chinese tertiary centers. Three expert and 4 novice endoscopists performed white-light endoscopy followed by LCE and HRME. All optical images were compared with the gold standard of histopathology. RESULTS By using a per-biopsy analysis, the sensitivity of LCE vs LCE + HRME was 96% vs 91% (P = .0832), specificity was 48% vs 88% (P < .001), positive predictive value was 22% vs 45% (P < .0001), negative predictive value was 98% vs 98% (P = .3551), and overall accuracy was 57% vs 90% (P < .001), respectively. By using a per-patient analysis, the sensitivity of LCE vs LCE + HRME was 100% vs 95% (P = .16), specificity was 29% vs 79% (P < .001), positive predictive value was 32% vs 60%, 100% vs 98%, and accuracy was 47% vs 83% (P < .001). With the use of HRME, 136 biopsies (60%; 95% confidence interval, 53%-66%) could have been spared, and 55 patients (48%; 95% confidence interval, 38%-57%) could have been spared any biopsy. CONCLUSIONS In this trial, HRME improved the accuracy of LCE for esophageal squamous cell neoplasia screening and surveillance. HRME may be a cost-effective optical biopsy adjunct to LCE, potentially reducing unnecessary biopsies and facilitating real-time decision making in globally underserved regions. ClinicalTrials.gov, NCT 01384708.

Low-cost high-resolution microendoscopy for the detection of esophageal squamous cell neoplasia

BACKGROUND & AIMS Esophageal squamous cell neoplasia has a high mortality rate as a result of late detection. In high-risk regions such as China, screening is performed by Lugols chromoendoscopy (LCE). LCE has low specificity, resulting in unnecessary tissue biopsy with a subsequent increase in procedure cost and risk. The purpose of this study was to evaluate the accuracy of a novel, low-cost, high-resolution microendoscope (HRME) as an adjunct to LCE. METHODS In this prospective trial, 147 consecutive high-risk patients were enrolled from 2 US and 2 Chinese tertiary centers. Three expert and 4 novice endoscopists performed white-light endoscopy followed by LCE and HRME. All optical images were compared with the gold standard of histopathology. RESULTS By using a per-biopsy analysis, the sensitivity of LCE vs LCE + HRME was 96% vs 91% (P = .0832), specificity was 48% vs 88% (P < .001), positive predictive value was 22% vs 45% (P < .0001), negative predictive value was 98% vs 98% (P = .3551), and overall accuracy was 57% vs 90% (P < .001), respectively. By using a per-patient analysis, the sensitivity of LCE vs LCE + HRME was 100% vs 95% (P = .16), specificity was 29% vs 79% (P < .001), positive predictive value was 32% vs 60%, 100% vs 98%, and accuracy was 47% vs 83% (P < .001). With the use of HRME, 136 biopsies (60%; 95% confidence interval, 53%-66%) could have been spared, and 55 patients (48%; 95% confidence interval, 38%-57%) could have been spared any biopsy. CONCLUSIONS In this trial, HRME improved the accuracy of LCE for esophageal squamous cell neoplasia screening and surveillance. HRME may be a cost-effective optical biopsy adjunct to LCE, potentially reducing unnecessary biopsies and facilitating real-time decision making in globally underserved regions. ClinicalTrials.gov, NCT 01384708.

In vivo classification of colorectal neoplasia using high-resolution microendoscopy: Improvement with experience.

High‐resolution microendoscopy (HRME) is a novel, low‐cost “optical biopsy” technology that allows for subcellular imaging. The study aim was to evaluate the learning curve of HRME for the differentiation of neoplastic from non‐neoplastic colorectal polyps.

Sa1827 Diagnostic Accuracy of a Novel Low Cost Microendoscope for the Detection of Barrett's Neoplasia: A Prospective, Single-Center Trial

istry (IHC). IECs were isolated and fractionated into cytosolic and nuclear fractions. p53 levels were assessed using WB and RT-PCR. Cleaved caspase-3 protein levels were assessed by WB. Target genes of p53 including survivin, p21, and Perp were measured using RTPCR. Results: WB of purified IECs 5 days after ICR revealed that nuclear p53 levels in the ICR mice were decreased compared to sham operated mice. Survivin, which is repressed by p53 transcription, was increased by RT-PCR (2-fold) and most dramatically by IHC with (3-5 fold) more survivin positive IEC staining through the lower to mid crypt regions in the ICR mice. We next examined other p53 target genes. Perp and p21 mRNA showed a 1.5 and 2 fold respective decreases in the RT-PCR of WT ICR as compared to sham surgery. Cleaved caspase-3 protein levels demonstrated a 2-fold decrease in the WT ICR as compared to sham surgery.Conclusions : The post-surgical epithelial responses after intestinal resection continue to be poorly understood. We demonstrated the importance of the down-regulation of p53 after ICR. As expected, survivin levels increased in the ICR mouse consistent with the notion that p53 repression and survivin induction contribute to the expanded surface area in ICR mice.

Diagnosis of neoplasia in Barrett's esophagus using vital-dye enhanced fluorescence imaging.

The ability to differentiate benign metaplasia in Barretts Esophagus (BE) from neoplasia in vivo remains difficult as both tissue types can be flat and indistinguishable with white light imaging alone. As a result, a modality that highlights glandular architecture would be useful to discriminate neoplasia from benign epithelium in the distal esophagus. VFI is a novel technique that uses an exogenous topical fluorescent contrast agent to delineate high grade dysplasia and cancer from benign epithelium. Specifically, the fluorescent images provide spatial resolution of 50 to 100 μm and a field of view up to 2.5 cm, allowing endoscopists to visualize glandular morphology. Upon excitation, classic Barretts metaplasia appears as continuous, evenly-spaced glands and an overall homogenous morphology; in contrast, neoplastic tissue appears crowded with complete obliteration of the glandular framework. Here we provide an overview of the instrumentation and enumerate the protocol of this new technique. While VFI affords a gastroenterologist with the glandular architecture of suspicious tissue, cellular dysplasia cannot be resolved with this modality. As such, one cannot morphologically distinguish Barretts metaplasia from BE with Low-Grade Dysplasia via this imaging modality. By trading off a decrease in resolution with a greater field of view, this imaging system can be used at the very least as a red-flag imaging device to target and biopsy suspicious lesions; yet, if the accuracy measures are promising, VFI may become the standard imaging technique for the diagnosis of neoplasia (defined as either high grade dysplasia or cancer) in the distal esophagus.