A single institution experience with the new bethesda system for reporting thyroid cytopathology: correlation with existing cytologic, clinical, and histological data.

Our goal was to evaluate the Bethesda system (TBS) in comparison to the previously used system at our institution. One hundred consecutive thyroid fine needle aspirations (FNAs) and 45 consecutive indeterminate FNAs were reviewed by two cytopathology-boarded pathologists, diagnosed based on TBS and correlated with management and follow-up. Re-evaluation led to a diagnosis change in 48% of cases. Thirty-nine percent of benign cases were unsatisfactory under TBS. For malignant diagnoses the positive predictive value (PPV) was unchanged, while the negative predictive value (NPV) was slightly improved using TBS. Both the PPV and NPV were improved for actionable diagnoses. Inter-observer variability across all categories was in moderate agreement. Clinical management of both follicular lesion (FL) and indeterminate cases ranged from none to immediate surgery. Repeat FNA resolved the diagnosis in 50% of indeterminate cases. Indeterminate cases had an overall malignancy rate of 27%; higher in pre- (46%) than post-TBS cases (8%). Inter-observer variability between the reviewing pathologists and the original pathologists for indeterminate cases was fair, and between the two reviewing pathologists was moderate. Using TBS criteria increased the unsatisfactory rate and led to improved prediction of malignancy and actionable diagnoses. The pre-Bethesda diagnosis of FL at our institution led to inconsistent clinical management. Clinical management of patients with indeterminate diagnoses was essentially unchanged following adoption of TBS. The moderate inter-observer agreement between the reviewing pathologists may be related to level of cytology experience, strict adherence to TBS, and the exclusive use of cytomorphology for diagnosis.

PLGA nanoparticle-mediated delivery of tumor antigenic peptides elicits effective immune responses.

The peptide vaccine clinical trials encountered limited success because of difficulties associated with stability and delivery, resulting in inefficient antigen presentation and low response rates in patients with cancer. The purpose of this study was to develop a novel delivery approach for tumor antigenic peptides in order to elicit enhanced immune responses using poly(DL-lactide-co-glycolide) nanoparticles (PLGA-NPs) encapsulating tumor antigenic peptides. PLGA-NPs were made using the double emulsion-solvent evaporation method. Artificial antigen-presenting cells were generated by human dendritic cells (DCs) loaded with PLGA-NPs encapsulating tumor antigenic peptide(s). The efficiency of the antigen presentation was measured by interferon-γ ELISpot assay (Vector Laboratories, Burlingame, CA). Antigen-specific cytotoxic T lymphocytes (CTLs) were generated and evaluated by CytoTox 96(®) Non-Radioactive Cytotoxicity Assay (Promega, Fitchburg, WI). The efficiency of the peptide delivery was compared between the methods of emulsification in incomplete Freund's adjuvant and encapsulation in PLGA-NPs. Our results showed that most of the PLGA-NPs were from 150 nm to 500 nm in diameter, and were negatively charged at pH 7.4 with a mean zeta potential of -15.53 ± 0.71 mV; the PLGA-NPs could be colocalized in human DCs in 30 minutes of incubation. Human DCs loaded with PLGA-NPs encapsulating peptide induced significantly stronger CTL cytotoxicity than those pulsed with free peptide, while human DCs loaded with PLGA-NPs encapsulating a three-peptide cocktail induced a significantly greater CTL response than those encapsulating a two-peptide cocktail. Most importantly, the peptide dose encapsulated in PLGA-NPs was 63 times less than that emulsified in incomplete Freund's adjuvant, but it induced a more powerful CTL response in vivo. These results demonstrate that the delivery of peptides encapsulated in PLGA-NPs is a promising approach to induce effective antitumor CTL responses in vivo.

Imaging of the interaction of cancer cells and the lymphatic system.

A thorough understanding of the lymphatic system and its interaction with cancer cells is crucial to our ability to fight cancer metastasis. Efforts to study the lymphatic system had previously been limited by the inability to visualize the lymphatic system in vivo in real time. Fluorescence imaging can address these limitations and allow for visualization of lymphatic delivery and trafficking of cancer cells and potentially therapeutic agents as well. Here, we review recent articles in which antibody-fluorophore conjugates are used to label the lymphatic network and fluorescent proteins to label cancer cells in the evaluation of lymphatic delivery and imaging.

Benign lymphoid hyperplasia (pseudolymphoma) of soft tissue.

Benign lymphoid hyperplasia (pseudolymphoma) has been reported in the skin, lungs, orbit, and gastrointestinal tract, but only rarely in soft tissues. These lesions mimic lymphoma both clinically and histologically. We describe a case of a pseudolymphoma of the deep soft tissues of the lower extremity. The lesion was composed of nonencapsulated lymphoid tissue with involvement of adjacent fat and connective tissues and multiple variably sized well-polarized germinal centers. Immunohistochemical staining, flow cytometry, chromogenic in situ hybridization for κ/λ light-chain restriction, and polymerase chain reaction for T- and B-cell gene rearrangements all revealed a polyclonal population of T and B cells, consistent with a benign reactive process. So far as we know, pseudolymphoma of the deep soft tissues has been described only once previously in the medical literature.

A unique case of an indolent CD56-positive T-cell lymphoproliferative disorder of the gastrointestinal tract: a lesion potentially misdiagnosed as natural killer/T-cell lymphoma.

Primary intestinal natural killer (NK)/T-cell lymphoma (nasal-type) and enteropathy-associated T-cell lymphoma, type II, are CD56-positive lymphoproliferative disorders with very poor survival rates. We report a long-surviving patient with a CD56-positive T-cell lymphoproliferative disorder of the gastrointestinal tract that presented as vomiting, diarrhea, weight loss, and pain. This patient was referred to the university hospital as a case of peripheral T-cell lymphoma due to this CD56-positive lymphocyte population. There was no evidence of enteropathy; and the infiltrates were negative for CD8, Epstein-Barr virus, and T-cell receptor gene rearrangement. Despite its persistence for 8 years, the clinical course has remained indolent. This report confirms that patients may rarely present with a CD56-positive NK/T-cell-like proliferation of the gastrointestinal tract, yet follow an indolent clinical course. Thus, all pathologic features of enteropathy-associated T-cell lymphoma or NK/T-cell lymphoma should be present before making this diagnosis and exposing the patient to toxic chemotherapy.

Disruption of angiogenesis and tumor growth with an orally active drug that stabilizes the inactive state of PDGFRbeta/B-RAF.

Kinases are known to regulate fundamental processes in cancer including tumor proliferation, metastasis, neovascularization, and chemoresistance. Accordingly, kinase inhibitors have been a major focus of drug development, and several kinase inhibitors are now approved for various cancer indications. Typically, kinase inhibitors are selected via high-throughput screening using catalytic kinase domains at low ATP concentration, and this process often yields ATP mimetics that lack specificity and/or function poorly in cells where ATP levels are high. Molecules targeting the allosteric site in the inactive kinase conformation (type II inhibitors) provide an alternative for developing selective inhibitors that are physiologically active. By applying a rational design approach using a constrained amino-triazole scaffold predicted to stabilize kinases in the inactive state, we generated a series of selective type II inhibitors of PDGFRbeta and B-RAF, important targets for pericyte recruitment and endothelial cell survival, respectively. These molecules were designed in silico and screened for antivascular activity in both cell-based models and a Tg(fli1-EGFP) zebrafish embryogenesis model. Dual inhibition of PDGFRbeta and B-RAF cellular signaling demonstrated synergistic antiangiogenic activity in both zebrafish and murine models of angiogenesis, and a combination of previously characterized PDGFRbeta and RAF inhibitors validated the synergy. Our lead compound was selected as an orally active molecule with favorable pharmacokinetic properties which demonstrated target inhibition in vivo leading to suppression of murine orthotopic tumors in both the kidney and pancreas.

Monotherapy with a tumor-targeting mutant of S. typhimurium inhibits liver metastasis in a mouse model of pancreatic cancer.

Cancer of the exocrine pancreas is the fourth leading cause of cancer deaths in the United States. Currently, surgical resection is the only hope for cure. The majority of patients present with locally-advanced or metastatic disease. The most common site for distant metastasis is the liver. We report here a modified auxotrophic strain of S. typhimurium that can target and inhibit the growth of liver metastasis in a mouse model of pancreatic cancer. This strain of S. typhimurium is auxotrophic (leucine-arginine dependent) but apparently receives sufficient nutritional support from tumor tissue. To increase tumor targeting ability and tumor killing efficacy, this strain was further modified by re-isolation from a tumor growing in a nude mouse and termed A1-R. In the present study, we demonstrate the efficacy of locally- as well as systemically-administered A1-R on liver metastasis of pancreatic cancer. Mice treated with A1-R given locally via intrasplenic injection or systemically via tail vein injection had a much lower hepatic and splenic tumor burden compared with control mice. Systemic treatment with intravenous A1-R also increased survival time. All results were statistically significant. This study suggests the clinical potential of bacterial treatment of a critical metastatic target of pancreatic cancer.

Case report: focal nesidioblastosis ("nesidioblastoma") in an adult.

Adult nesidioblastosis is an uncommon cause of hyperinsulinemic hypoglycemia characterized by diffuse islet hyperplasia with beta-cell hypertrophy and atypia. The cause of nesidioblastosis in adults is unclear but may be different from nesidioblastosis in infants. In contrast to infants, a focal form of adult nesidioblastosis (ie, "nesidioblastoma") has not been documented, although proposed. We report a 44-year-old man with symptomatic hypoglycemia and localized nesidioblastosis treated with surgical enucleation resulting in normalization of blood glucose. Postoperative euglycemia has persisted in this patient to date (4 months at the time of manuscript submission).

Upregulation of thrombospondin-1 and angiogenesis in an aggressive human pancreatic cancer cell line selected for high metastasis.

Pancreatic cancer remains a leading cause of death despite its relatively low incidence. As in many other solid tumors, angiogenesis is critical to the growth and metastasis of this cancer. Through serial in vivo passages in mice, we have developed a highly aggressive variant of human pancreatic cancer cell line XPA-1 which shows more rapid primary tumor growth, faster time to metastasis, and more rapid lethality than the parental cell line. The high-metastatic variant developed a much denser tumor vasculature early during growth within the pancreas. Interestingly, examination of the in vitro growth of this aggressive variant yielded no significant difference from the parental cell line. Real-time PCR evaluation of genes involved in angiogenesis revealed a 24-fold increase in Thrombospondin-1 expression in cells derived from the high-metastatic variant when compared with the parental cell line. These findings provide direct evidence that elevated capability for angiogenesis, mediated by specific changes in gene expression, can lead to a large increase in cancer aggressiveness and resulting metastasis. These findings have important implications for the treatment of metastatic disease.

Extended-working-distance multiphoton micromanipulation microscope for deep-penetration imaging in live mice and tissue.

We constructed a multiphoton (2-P) microscope with space to mount and operate microphysiology hardware, and still acquire high quality 2-P images of tumor cells deep within tissues of live mice. We reconfigured for nondescanned 2-P imaging, a dedicated electrophysiology microscope, the Nikon FN1. This microscope is compact, with retractable objectives, allowing more stage space. The instrument is fitted with long-working-distance objectives (2.5- to 3.5-mm WD) with a narrow bore, high NA, and efficient UV and IR light transmission. The system is driven by a powerful 3.5-W peak power pulsed Ti-sapphire laser with a broad tuning range. This 2-P system images a fluorescent standard to a depth of 750 to 800 microm, acquires images of murine pancreatic tumors in vivo, and also images fluorescently labeled T-cells inside live, externalized mouse lymph nodes. Effective imaging depths range between 100 and 500 microm. This compares favorably with the 100- to 300 microm micron depth attained by many 2-P systems, especially descanned 2-P instruments, and 40-microm-deep imaging with confocal microscopes. The greater depth penetration is attributable to the use of high-NA long-working-distance water-dipping lenses incorporated into a nondescanned instrument with carefully configured laser beam introduction and image-acquisition optics. Thus the new system not only has improved imaging capabilities, but allows micromanipulation and maintenance of tissues and organs.

Fluorescent LYVE-1 antibody to image dynamically lymphatic trafficking of cancer cells in vivo.

BACKGROUND: The lymphatic system is a major route for cancer cell dissemination, and a potential target for antitumor therapy. Despite ongoing interest in this area of research, the real-time behavior of cancer cells trafficking in the lymphatic system is poorly understood due to lack of appropriate tools to image this process. MATERIALS AND METHODS: We have used monoclonal-antibody and fluorescence technology to color-code lymphatic vessels and the cancer cells inside them in a living animal. Monoclonal anti-mouse LYVE-1 antibody was conjugated to a green fluorophore and delivered to the lymphatic system of a nude mouse, allowing imaging of mouse lymphatics. Tumor cells engineered to express red fluorescent protein were then imaged traveling within the labeled lymphatics in real time. RESULTS: AlexaFluor-labeled monoclonal anti-mouse LYVE-1 created a durable signal with clear delineation of lymphatic architecture. The duration of fluorescent signal after conjugated LYVE-1 delivery was far superior to that of fluorescein isothiocyanate-dextran or control fluorophore-conjugated IgG. Tumor cells engineered to express red fluorescent protein delivered to the inguinal lymph node enabled real-time tracking of tumor cell movement within the green fluorescent-labeled lymphatic vessels. CONCLUSIONS: This technology offers a powerful tool for the in vivo study of real-time trafficking of tumor cells within lymphatic vessels, for the deposition of the tumor cells in lymph nodes, as well as for screening of potential antitumor lymphatic therapies.

Chapter 2. Color-coded fluorescent mouse models of cancer cell interactions with blood vessels and lymphatics.

Several new strategies now exist for imaging cancer cell interactions with both blood vessels and lymphatics in living animals. Tumors labeled with fluorescent proteins allow the nonluminous capillaries and larger blood vessels to be clearly visualized against the bright tumor fluorescence via either intravital or whole-body imaging. Signal attenuation by overlying tissue can be markedly reduced by opening a reversible skin flap in the light path, increasing detection sensitivity. With this increase in observable depth of tissue, many previously obscured small tumor vessels can be imaged. In addition, dual-color fluorescence imaging, effected by using red fluorescent protein (RFP)-expressing tumors growing in green fluorescent protein (GFP)-expressing transgenic mice, can show with great clarity tumor-stroma interactions, including the developing tumor vasculature. The GFP-expressing host vasculature, both mature and nascent, can be distinguished from the RFP-expressing tumor itself in this model. Transgenic mice with GFP gene expression driven by the nestin promoter offer another way to image the developing tumor vasculature. In this model system, only nascent blood vessels express GFP, allowing newly developing blood vessels to be imaged against a background of RFP-expressing tumor cells. Finally, dual-color imaging technology can facilitate the imaging of cancer cell interactions with lymphatics. Delivery of FITC-dextran or fluorescent antibodies specific for lymphatic endothelium to the lymphatics around an RFP-expressing tumor allows imaging of tumor cell shedding into the lymphatic system. This imaging technology has the potential to visualize each step of tumor progress.

Color-coded imaging of splenocyte-pancreatic cancer cell interactions in the tumor microenvironment.

In spite of advances in surgical and medical care, pancreatic cancer remains a leading cause of cancer-related death in the United States. An understanding of cancer-cell interactions with host cells is critical to our ability to develop effective antitumor therapeutics for pancreatic cancer. We report here a color-coded model system for imaging cancer cell interactions with host immune cells within the native pancreas. A human pancreatic cancer cell line engineered to express green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) (DsRed2) in the cytoplasm was orthotopically implanted into the pancreas of a nude mouse. After 10-14 days, red or green fluorescent splenocytes from immune-competent transgenic-mouse donors expressing RFP and GFP, respectively, were delivered systemically to the pancreatic cancer-bearing nude mice. Animals were imaged after splenocyte delivery using high-resolution intravital imaging systems. At 1 day after iv injection red- or green-fluorescent spleen cells were found distributed in lung, liver, spleen and pancreas. By 4 days after cell delivery, however, the immune cells could be clearly imaged surrounding the tumor cells within the pancreas as well as collecting within lymphatic tissues such as lymph nodes and spleen. With the high-resolution intravital imaging afforded by the Olympus IV100 and OV100 systems, the interactions of the dual-colored cancer cells and the red- or green-fluorescent spleen cells could be clearly imaged in this orthotopic pancreatic cancer model. This color-coded in vivo imaging technology offers a novel approach to imaging the interactions of cancer and immune cells in the tumor microenvironment (TME).

Fluorophore-conjugated anti-CEA antibody for the intraoperative imaging of pancreatic and colorectal cancer.

INTRODUCTION: Colorectal and pancreatic cancers together comprise the third and fourth most common causes of cancer-related death in the United States. In both of these cancers, complete detection of primary and metastatic lesions at the time of surgery is critical to optimal surgical resection and appropriate patient treatment. MATERIALS AND METHODS: We have investigated the use of fluorophore-labeled anti-carcinoembryonic antigen (CEA) monoclonal antibody to aid in cancer visualization in nude mouse models of human colorectal and pancreatic cancer. Anti-CEA was conjugated with a green fluorophore. Subcutaneous, orthotopic primary and metastatic human pancreatic and colorectal tumors were easily visualized with fluorescence imaging after administration of conjugated anti-CEA. The fluorescence signal was detectable 30 min after systemic antibody delivery and remained present for 2 weeks, with minimal in vivo photobleaching after exposure to standard operating room lighting. Tumor resection techniques revealed improved ability to resect labeled tumor tissue under fluorescence guidance. Comparison of two different fluorophores revealed differences in dose-response and photobleaching in vivo. CONCLUSION: These results indicate that fluorophore-labeled anti-CEA offers a novel intraoperative imaging technique for enhanced visualization of tumors in colorectal and pancreatic cancer when CEA expression is present, and that the choice of fluorophore significantly affects the signal intensity in the labeled tumor.

Imaging of primary and metastatic pancreatic cancer using a fluorophore-conjugated anti-CA19-9 antibody for surgical navigation.

BACKGROUND: Despite recent surgical advances, pancreatic cancer remains the fourth leading cause of cancer-related death in the United States. This is due to inaccurate staging and difficulty in achieving negative margins at the time of pancreaticoduodenectomy. CA19-9 is a carbohydrate tumor-associated antigen found in up to 94% of pancreatic adenocarcinomas. In this study we investigate the use of a fluorophore-labeled anti-CA19-9 monoclonal antibody to improve intraoperative visualization of both primary and metastatic tumors in a mouse model of pancreatic cancer. METHODS: A monoclonal antibody specific for CA19-9 was conjugated to a green fluorophore and delivered to tumor-bearing mice as a single intravenous (IV) dose. Intravital fluorescence imaging was used to localize tumor implants 24 h after antibody administration. RESULTS: Using fluorescence imaging, the primary tumor was clearly visible at laparotomy, as were small metastatic implants within the liver and spleen and on the peritoneum. These tumor implants, which were nearly impossible to see using standard bright-field imaging, demonstrated clear fluorescence under LED light excitation. The fluorescence signal within the tumor tissue was maintained for over 3 weeks after a single administration of the labeled antibody. Histologic evaluation of tissue from animals treated with the conjugated anti-CA19-9 antibody likewise revealed strong staining of the tumor cells with minimal background staining of the peritumoral stroma. CONCLUSIONS: Fluorophore-labeled anti-CA19-9 offers a novel intraoperative imaging technique for enhanced visualization of primary and metastatic tumors in pancreatic cancer when CA19-9 expression is present and may improve intraoperative staging and efficacy of resection.

The efficacy of breast MRI in predicting breast conservation therapy.

BACKGROUND: Breast conservation therapy (BCT) has equal efficacy compared to mastectomy in treating breast cancer. Accurate pre-operative measurement of tumor size can limit re-excision procedures. Breast MRI may improve pre-operative evaluation of extent of disease. OBJECTIVE: To examine the correlation of extent of disease on breast MRI with pathologic data to determine the utility of breast MRI in surgical planning of BCT. METHODS: We retrospectively reviewed our prospective database of women undergoing breast MRI. We identified 115 women with breast cancer who underwent a breast MRI and a surgical resection from 2000 to 2003. We compared patients with high-grade tumors (HG, n = 40) to patients with low grade (LG, n = 75). RESULTS: The size of the tumor on MRI correlated with the pathologic size for HG tumors (HG R = 0.76 vs. LG R = 0.45, P = 0.033). Mastectomy was performed in 53 patients. In 10 patients with LG tumors, the MRI findings overestimated their disease. In 11 out of 115 patients the primary tumor or a second tumor was only seen by MRI. CONCLUSION: Breast MRI does change surgical management by detecting additional malignancies. Breast MRI is accurate in staging extent of disease in the breast in patients with HG tumors.