Sunil Singhal

University of Pennsylvania, Filadelfia, Pennsylvania, United States

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Publications (98)431.67 Total impact

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    ABSTRACT: Background and objectives: Current methods of intraoperative breast cancer margin assessment are labor intensive, not fully reliable, and time consuming; therefore novel strategies are necessary. We hypothesized that near infrared (NIR) intraoperative molecular imaging using systemic indocyanine green (ICG) would be helpful in discerning tumor margins. Methods: A mammary cancer cell line, 4T1, was used to establish tumors in mouse flanks (n = 60). Tumors were excised 24 hr after intravenous ICG. Assessment of residual tumor in the wound bed was performed using a combination of NIR imaging and traditional method (by visual inspection and palpation) versus traditional method alone. Next we performed a clinical trial to evaluate the role of NIR imaging after systemic ICG for the margin assessment of 12 patients undergoing breast-conserving surgery. Results: Traditional margin assessment identified 30% of positive margins while NIR imaging identified 90% of positive margins. In our clinical trial, all tumors were detected by NIR imaging and there was fluorescent evidence of residual tumor in the tumor bed in 6 of the 12 patients. None of these patients had positive margins on pathology. Conclusions: Systemic ICG reliably accumulates in breast cancers in murine models as well as human breast cancer. While NIR imaging is helpful for detection of retained tumor margins in our animal model, intraoperative imaging for precise margin detection will need further refinement before clinical value can be obtained. J. Surg. Oncol. © 2016 Wiley Periodicals, Inc.
    No preview · Article · Feb 2016 · Journal of Surgical Oncology
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    ABSTRACT: Positive margins following pulmonary resection of non-small cell lung cancer (NSCLC) occur in approximately 5-15% of patients undergoing a curative procedure. The presence of positive margins negatively impacts long-term outcomes by setting the stage for local and potentially distant disease recurrence. Despite major clinical ramifications, there are very few dedicated reports that examine the implications of positive margins following surgery for NSCLC. Furthermore, published series are typically retrospective studies from single institutions. In this review we analyze published data with special consideration of four pertinent questions: (i) what are the long term outcomes of a positive margin following pulmonary resection?, (ii) is intraoperative margin assessment by frozen section reliable?, (iii) what is the optimal distance of the tumor margin to the surgical margin?, and (iv) should adjuvant chemotherapy and/or radiation therapy be used in the setting of a positive surgical margin? J. Surg. Oncol. © 2015 Wiley Periodicals, Inc.
    No preview · Article · Dec 2015 · Journal of Surgical Oncology
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    ABSTRACT: Near infrared (NIR) molecular imaging is useful to identify tumor margins during surgery; however, the value of this technology has not been evaluated for tumors that have been pre-treated with chemotherapy. We hypothesized that NIR molecular imaging could locate mediastinal tumor margins in a murine model after neoadjuvant chemotherapy. Flank thymomas were established on mice. Two separate experiments were performed for tumor margin detection. The first experiment compared (i) surgery and (ii) surgery + NIR imaging. The second experiment compared (iii) preoperative chemotherapy + surgery, and (iv) preoperative chemotherapy + surgery + NIR imaging. NIR imaging occurred following systemic injection of indocyanine green. Margins were assessed for residual tumor cells by pathology. NIR imaging was superior at detecting retained tumor cells during surgery compared to standard techniques (surgery alone vs. surgery + NIR imaging, 20% vs. 80%, respectively). Following chemotherapy, the sensitivity of NIR imaging of tumor margins was not significantly altered. The mean in vivo tumor-to-background fluorescence ratio was similar in the treatment-naïve and chemotherapy groups ((p = 0.899): 3.79 ± 0.69 (IQR 3.29 - 4.25) vs. 3.79 ± 0.52 (IQR 3.40 - 4.03)). We conclude that chemotherapy does not affect tumor fluorescence or identification of retained cancer cells at margins.
    No preview · Article · Dec 2015 · Oncotarget
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    ABSTRACT: Intraoperative optical cancer imaging is an emerging technology in which surgeons employ fluorophores to visualize tumors, identify tumor-positive margins and lymph nodes containing metastases. This study compares instrumentation to measure tumor fluorescence. Three imaging systems (Spectropen, Glomax, Flocam) measured and quantified fluorescent signal-to-background ratios (SBR) in vitro, murine xenografts, tissue phantoms and clinically. Evaluation criteria included the detection of small changes in fluorescence, sensitivity of signal detection at increasing depths and practicality of use. In vitro, spectroscopy was superior in detecting incremental differences in fluorescence than luminescence and digital imaging (Ln[SBR] = 6.8 ± 0.6, 2.4 ± 0.3, 2.6 ± 0.1, p = 0.0001). In fluorescent tumor cells, digital imaging measured higher SBRs than luminescence (6.1 ± 0.2 vs. 4.3 ± 0.4, p = 0.001). Spectroscopy was more sensitive than luminometry and digital imaging in identifying murine tumor fluorescence (SBR = 41.7 ± 11.5, 5.1 ± 1.8, 4.1 ± 0.9, p = 0.0001), and more sensitive than digital imaging at detecting fluorescence at increasing depths (SBR = 7.0 ± 3.4 vs. 2.4 ± 0.5, p = 0.03). Lastly, digital imaging was the most practical and least time-consuming. All methods detected incremental differences in fluorescence. Spectroscopy was the most sensitive for small changes in fluorescence. Digital imaging was the most practical considering its wide field of view, background noise filtering capability, and sensitivity to increasing depth.
    Full-text · Article · Nov 2015 · Scientific Reports
  • Sunil Singhal · John C. Kucharczuk
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    ABSTRACT: Address reprint requests to John Kucharczuk, MD, Department of Surgery, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA.
    No preview · Article · Nov 2015 · Operative Techniques in Thoracic and Cardiovascular Surgery
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    ABSTRACT: Background. Intraoperative imaging can identify cancer cells in order to improve resection; thus fluorescent contrast agents have emerged. Our objective was to do a preclinical comparison of two fluorescent dyes, EC17 and OTL38, which both target folate receptor but have different fluorochromes. Materials. HeLa and KB cells lines were used for in vitro and in vivo comparisons of EC17 and OTL38 brightness, sensitivity, pharmacokinetics, and biodistribution. In vivo experiments were then performed in mice. Results. The peak excitation and emission wavelengths of EC17 and OTL38 were 470/520 nm and 774/794 nm, respectively. In vitro, OTL38 required increased incubation time compared to EC17 for maximum fluorescence; however, peak signal-to-background ratio (SBR) was 1.4-fold higher compared to EC17 within 60 minutes (p < 0.001). Additionally, the SBR for detecting smaller quantity of cells was improved with OTL38. In vivo, the mean improvement in SBR of tumors visualized using OTL38 compared to EC17 was 3.3 fold (range 1.48-5.43). Neither dye caused noticeable toxicity in animal studies. Conclusions. In preclinical testing, OTL38 appears to have superior sensitivity and brightness compared to EC17. This coincides with the accepted belief that near infrared (NIR) dyes tend to have less autofluorescence and scattering issues than visible wavelength fluorochromes.
    Full-text · Article · Oct 2015

  • No preview · Article · Oct 2015 · Journal of the American College of Surgeons

  • No preview · Article · Oct 2015
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    ABSTRACT: Background: With increasing use of chest computed tomography scans, indeterminate pulmonary nodules are frequently detected as an incidental finding and present a diagnostic challenge. Tissue biopsy followed by histological review and immunohistochemistry is the gold standard to obtain a diagnosis and the most common malignant finding is a primary lung adenocarcinoma. Our objective was to determine whether an intraoperative optical biopsy (molecular imaging) may provide an alternative approach for determining if a pulmonary nodule is a primary lung adenocarcinoma. Methods: Before surgery, 30 patients with an indeterminate pulmonary nodule were intravenously administered a folate receptor-targeted fluorescent contrast agent specific for primary lung adenocarcinomas. During surgery, the nodule was removed and the presence of fluorescence (optical biopsy) was assessed in the operating room to determine if the nodule was a primary pulmonary adenocarcinoma. Standard-of-care frozen section and immunohistochemical staining on permanent sections were then performed as the gold standard to validate the results of the optical biopsy. Results: Optical biopsies identified 19 of 19 (100%) primary pulmonary adenocarcinomas. There were no false positive or false negative diagnoses. An optical biopsy required 2.4 minutes compared to 26.5 minutes for frozen section (P < 0.001) and it proved more accurate than frozen section in diagnosing lung adenocarcinomas. Conclusions: An optical biopsy has excellent positive predictive value for intraoperative diagnosis of primary lung adenocarcinomas. With refinement, this technology may prove to be an important supplement to standard pathology for examining close surgical margins, identifying lymph node involvement, and determining whether suspicious nodules are malignant.
    No preview · Article · Sep 2015 · Annals of surgery
  • Sunil Singhal

    No preview · Article · Sep 2015 · The Journal of thoracic and cardiovascular surgery
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    ABSTRACT: Myeloid derived suppressor cells (MDSCs) are an immunosuppressive population of immature myeloid cells found in advanced stage cancer patients and mouse tumor models. Production of inducible nitric oxide synthase (iNOS) and arginase, as well as other suppressive mechanisms, allow MDSCs to suppress T cell-mediated tumor clearance and foster tumor progression. Using an unbiased global gene expression approach in conditional p120-catenin knockout mice (L2-cre;p120ctnf/f), a model of oral-esophageal cancer, we have identified CD38 as playing a vital role in MDSC biology, previously unknown. CD38 belongs to the ADP-ribosyl cyclase family and possesses both ectoenzyme and receptor functions. It has been described to function in lymphoid and early myeloid cell differentiation, cell activation and neutrophil chemotaxis. We find that CD38 expression in MDSCs is evident in other mouse tumor models of esophageal carcinogenesis, and CD38high MDSCs are more immature than MDSCs lacking CD38 expression, suggesting a potential role for CD38 in the maturation halt found in MDSC populations. CD38high MDSCs also possess a greater capacity to suppress activated T cells, and promote tumor growth to a greater degree than CD38low MDSCs, likely as a result of increased iNOS production. Additionally, we have identified novel tumor-derived factors, specifically IL-6, IGFBP-3 and CXCL16, which induce CD38 expression by MDSCs ex vivo. Finally, we have detected an expansion of CD38-positive MDSCs in peripheral blood of advanced stage cancer patients and validated targeting CD38 in vivo as a novel approach to cancer therapy. Copyright © 2015, American Association for Cancer Research.
    No preview · Article · Aug 2015 · Cancer Research
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    ABSTRACT: Surgery is the most effective method to cure patients with solid tumors. New techniques in near-infrared (NIR) cancer imaging are being used to identify surgical margins and residual tumor cells in the wound. Our goal was to determine the optimal time and dose for imaging solid tumors using Indocyanine Green. Syngeneic murine flank tumor models were used to test NIR imaging of ICG at various doses ranging from 0 to 10 mg/kg. Imaging was performed immediately after injection and up to 72 hours later. Biodistribution in the blood and murine organs were quantified by spectroscopy and fluorescence microscopy. Based on these results, a six patient dose titration study was performed. In murine flank tumors, the tumor-to-background ratio (TBR) for ICG at doses less than 5 mg/kg were less than 2 fold at all time points, and the surgeons could not subjectively identify tissue contrast. However, for doses ranging from 5 mg/kg to 10 mg/kg, the TBR ranged from 2.1 to 8.0. The tumor signal was best appreciated at 24 hours and the background was least pronounced after 24 hours. Biodistribution studies in the blood and murine organs revealed excretion through the biliary tree and gastrointestinal tract, with minimal blood fluorescence at the higher doses. A follow up pilot study confirmed that these findings were applicable to lung cancer patients, and tumor was clearly delineated from surrounding normal tissue by NIR imaging. For non-hepatic solid tumors, we found ICG was optimal when dosed at 5 mg/kg and 24 hours before surgery.
    No preview · Article · Aug 2015 · American Journal of Nuclear Medicine and Molecular Imaging

  • No preview · Article · Aug 2015 · Cancer Research
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    ABSTRACT: During lung surgery, identification of surgical margins is challenging. We hypothesized that molecular imaging with a fluorescent probe to pulmonary adenocarcinomas could enhance residual tumor during resection. Mice with flank tumors received a contrast agent targeting folate receptor alpha. Optimal dose and time of injection was established. Margin detection was compared using traditional methods versus molecular imaging. A pilot study was then performed in three humans with lung adenocarcinoma. The peak tumor-to-background ratio (TBR) of murine tumors was 3.9. Fluorescence peaked at 2 h and was not improved beyond 0.1 mg/kg. Traditional inspection identified 30 % of mice with positive margins. Molecular imaging identified an additional 50 % of residual tumor deposits (p < 0.05). The fluorescent probe visually enhanced all human tumors with a mean TBR of 3.5. Molecular imaging is an important adjunct to traditional inspection to identify surgical margins after tumor resection.
    No preview · Article · Jul 2015 · Molecular imaging and biology: MIB: the official publication of the Academy of Molecular Imaging
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    ABSTRACT: Surgery is the most effective method to cure patients with solid tumors, and 50% of all cancer patients undergo resection. Local recurrences are due to tumor cells remaining in the wound, thus we explore near-infrared (NIR) fluorescence spectroscopy and imaging to identify residual cancer cells after surgery. Fifteen canines and two human patients with spontaneously occurring sarcomas underwent intraoperative imaging. During the operation, the wounds were interrogated with NIR fluorescence imaging and spectroscopy. NIR monitoring identified the presence or absence of residual tumor cells after surgery in 14/15 canines with a mean fluorescence signal-to-background ratio (SBR) of ∼16 . Ten animals showed no residual tumor cells in the wound bed (mean SBR<2 , P<0.001 ). None had a local recurrence at >1-year follow-up. In five animals, the mean SBR of the wound was >15 , and histopathology confirmed tumor cells in the postsurgical wound in four/five canines. In the human pilot study, neither patient had residual tumor cells in the wound bed, and both remain disease free at >1.5-year follow up. Intraoperative NIR fluorescence imaging and spectroscopy identifies residual tumor cells in surgical wounds. These observations suggest that NIR imaging techniques may improve tumor resection during cancer operations.
    No preview · Article · Jul 2015 · Journal of Biomedical Optics
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    ABSTRACT: Recent studies suggest that immunotherapy may offer a promising treatment strategy for early-stage malignant pleural mesothelioma (MPM), but advanced tumor burden may limit the efficacy of immunotherapy. Therefore, we hypothesized that surgical cytoreduction could restore the efficacy of vaccine-based immunotherapy for MPM. We developed a murine model of MPM through transduction of a mesothelioma cell line with mesothelin. We used this model to evaluate the efficacy of a Listeria monocytogenes vaccine expressing mesothelin. Tumor growth was significantly inhibited at four weeks in animals vaccinated two weeks prior to tumor cell inoculation as compared to those given an empty vector control (1371±420mm(3) versus 405±139mm(3); p<0.01). Mice vaccinated one week prior to tumor challenge also displayed significant reduction in tumor volume (1227±406mm(3) versus 309±173mm(3); p<0.01). The vaccine had no effect when administered concurrently with tumor challenge, or after tumors were established. Flow cytometry showed reduced mesothelin expression in large tumors, as well as tumor-associated immunosuppression due to increased myeloid derived suppressor cells (MDSCs). These factors may have limited vaccine efficacy for advanced disease. Surgical cytoreduction of established tumors restored the antitumor potency of the therapeutic vaccine, with significantly reduced tumor burden at post-operative day 18 (397±103mm(3) versus 1047±258mm(3); p<0.01). We found that surgery reduced MDSCs to levels comparable to those in tumor-naïve mice. This study demonstrates that cytoreduction surgery restores the efficacy of cancer vaccines for MPM by reducing tumor-related immunosuppression that impairs immunotherapy. Copyright © 2015. Published by Elsevier B.V.
    No preview · Article · May 2015 · Immunology letters
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    ABSTRACT: More than 80,000 people undergo resection of a pulmonary tumor each year, and the only method to determine if the tumor is malignant is histologic analysis. We propose that a targeted molecular contrast agent could bind lung adenocarcinomas, which could be identified using real-time optical imaging at the time of surgery. Fifty patients with a biopsy-proven lung adenocarcinoma were enrolled. Before surgery, patients were systemically administered 0.1 mg/kg of a fluorescent folate receptor alpha (FRα)-targeted molecular contrast agent by intravenous infusion. During surgery, tumors were imaged in situ and ex vivo, after the lung parenchyma was dissected to directly expose the tumor to the imaging system. Tumors ranged from 0.3 to 7.5 cm (mean: 2.6 cm), and 46 of 50 (92%) lung adenocarcinomas were fluorescent. No false uptake occurred, and in 2 cases, intraoperative imaging revealed tumor metastases (3 mm and 6 mm) that were not recognized preoperatively. Four adenocarcinomas were not fluorescent, and immunohistochemistry showed that these adenocarcinomas did not express FRα. Tumor fluorescence was independent of nodule size, uptake of 2-deoxy-2-((18)F)fluoro-D-glucose, histology, and tumor differentiation. Molecular imaging could identify only 7 of the 50 adenocarcinomas in situ in the patient without bisection. The most important predictor of the success of molecular imaging in locating the tumor in situ was the distance of the nodule from the pleural surface. Intraoperative molecular imaging with a targeted contrast agent can identify lung adenocarcinomas, and this technology is currently useful in patients with subpleural tumors, irrespective of size. With further refinements, this tool may prove useful in locating adenocarcinomas that are deeper in the lung parenchyma, in lymph nodes, and at pleural and resection margins. Copyright © 2015 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.
    No preview · Article · May 2015 · The Journal of thoracic and cardiovascular surgery
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    ABSTRACT: Background: Margin status can often be difficult to assess intraoperatively, particularly during partial nephrectomy given the time constraints related to renal hilar clamping. We hypothesized that a targeted molecular imaging approach could be used during surgery to identify tumor margins and confirm disease clearance. Methods: A novel tracer targeting FRα (EC17) was used in murine models of RCC to identify positive margins after surgery. Positive margins were detected due to elevated tumor-to-background ratios (TBR) of the tumor to surrounding normal tissues. We conducted a pilot study on 4 human patients using EC17 prior to surgery, with intraoperative imaging during the operation. Results: Renal tumors were harvested from the operating room and confirmed FRα is highly expressed on 65% of clear cell RCC. In murine models, intraoperative imaging of RCC revealed TBR of 8.2 ± 1.1 for RCC10, 11.2 ± 1.1 for 786-0 and 4.3 ± 1.1 for UMRC2. Intraoperative imaging of the surgical resection bed identified residual disease in 24% more animals than visual inspection. In the human pilot study, targeted molecular imaging identified 2 out of 4 RCC, and had no false positives. In these 2 cases, the TBR was 3.7 and 4.6. In both cases, we confirmed disease clearance, and tumor fluorescence did not correlate with nodule size or tumor grade. Conclusions: This is the first-in-human demonstration of identifying RCC during surgery by using a targeted molecular contrast agent. This approach may lead to superior method to identify malignancy and tumor borders in the intraoperative setting.
    No preview · Article · Apr 2015 · The Journal of Urology
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    ABSTRACT: Over-expression of folate receptor alpha on cancer cells has been frequently exploited for delivery of folate-targeted imaging and therapeutic agents to tumors. Because limited information exists on expression of the beta isoform of the folate receptor in human cancers (FR-β), we have evaluated the immunohistochemical staining pattern of FR-β in 992 tumor sections from 20 different human cancer types using a new anti-human FR-β monoclonal antibody. FR-β expression was shown to be more pronounced in cells within the stroma, primarily macrophages and macrophage-like cells than cancer cells in every cancer type studied. Moreover, FR-β expression in both cancer and stromal cells was found to be statistically more prominent in females than males. A significant positive correlation was also observed between FR-β expression on stromal cells and both the stage of the cancer and the presence of lymph node metastases. Based on these data we conclude FR-β may constitute a good target for specific delivery of therapeutic agents to activated macrophages and that accumulation of FR-β positive macrophages in the stroma could serve as a useful indicator of a tumor's metastatic potential.
    Full-text · Article · Mar 2015 · Oncotarget
  • Jane J Keating · Gregory T Kennedy · Sunil Singhal

    No preview · Article · Mar 2015 · The Journal of thoracic and cardiovascular surgery

Publication Stats

2k Citations
431.67 Total Impact Points


  • 2005-2015
    • University of Pennsylvania
      • Department of Surgery
      Filadelfia, Pennsylvania, United States
  • 2004-2015
    • William Penn University
      Filadelfia, Pennsylvania, United States
  • 2002-2015
    • Hospital of the University of Pennsylvania
      • • Division of Cardiothoracic Surgery
      • • Department of Surgery
      Filadelfia, Pennsylvania, United States
  • 2014
    • Emory University
      • Department of Chemistry
      Atlanta, Georgia, United States
  • 2013-2014
    • Treatment Research Institute, Philadelphia PA
      Filadelfia, Pennsylvania, United States
  • 2012
    • University of Texas Southwestern Medical Center
      • Medical School
      Dallas, Texas, United States
  • 2003-2006
    • Johns Hopkins University
      • • Department of Surgery
      • • Department of Medicine
      Baltimore, Maryland, United States