Charles R. Handorf

MicroRNA miR-21 Regulates the Metastatic Behavior of B16 Melanoma Cells

Background: miRNAs are involved in many critical biological processes. Results: miR-21 induction is STAT3-dependent, and miR-21 knockdown inhibited melanoma cell proliferation and migration and enhanced apoptosis. Although B16 cells produced large lung metastases in mice, miR-21 knockdown cells only formed small lung lesions. Conclusion: miR-21 regulates the metastatic behavior of melanoma. Significance: miR-21 is identified as a potential drug target in melanoma. MicroRNA-21 (miR-21) is overexpressed in many human tumors and has been linked to various cellular processes altered in cancer. miR-21 is also up-regulated by a number of inflammatory agents, including IFN, which is of particular interest considering the close relationship between inflammation and cancer. Because miR-21 appears to be overexpressed in human melanoma, we examined the role of miR-21 in cancer development and metastasis in B16 mouse melanoma cells. We found that miR-21 is a member of an IFN-induced miRNA subset that requires STAT3 activation. To characterize the role of miR-21 in melanoma behavior, we transduced B16 cells with lentivirus encoding a miR-21 antagomir and isolated miR-21 knockdown B16 cells. miR-21 knockdown or IFN treatment alone inhibited B16 cell proliferation and migration in vitro, and in combination they had an enhanced effect. Moreover, miR-21 knockdown sensitized B16 cells to IFN-induced apoptosis. In B16 cells miR-21 targeted tumor suppressor (PTEN and PDCD4) and antiproliferative (BTG2) proteins. To characterize the role of miR-21 in vivo, empty vector- and antagomiR-21-transduced B16 melanoma cells were injected via tail vein into syngeneic C57BL/6 mice. Although empty vector-transduced B16 cells produced large lung metastases, miR-21 knockdown cells only formed small lung lesions. Importantly, miR-21 knockdown tumor-bearing mice exhibited prolonged survival compared with empty vector tumor-bearing mice. Thus, miR-21 regulates the metastatic behavior of B16 melanoma cells by promoting cell proliferation, survival, and migration/invasion as well as by suppressing IFN action, providing important new insights into the role of miR-21 in melanoma.

Small Intestinal Submucosa for Vascular Reconstruction in the Presence of Gastrointestinal Contamination

Introduction:Surgical options for vascular reconstruction in a contaminated field are limited and include prosthetic reconstruction or ligation with extra-anatomic bypass. With prosthetic insertion, rates of graft infection and failures (pseudoaneurysms and thrombosis) are high. In the emergent situations, extra-anatomic bypass is time-consuming and complex, and it produces marginal long-term results. Small intestinal submucosa (SIS) is a cell-free collagen matrix derived from porcine small intestine. Preliminary studies have demonstrated its ability to be remodeled into host tissue. In this study, we compared SIS to polytetrafluoroethylene (PTFE) as a vascular patch for arterial repair in the presence of massive gastrointestinal contamination to evaluate graft patency, incorporation, infection, and aneurysm formation. Methods:Adult mongrel pigs underwent general anesthesia with Isoflurane and were then randomized to 1 of 3 groups: control, contamination (colon puncture with stool contamination of the pelvis), or shock + contamination (40% blood volume for 1 hour, then resuscitation with shed blood and crystalloid, plus contamination). All groups then underwent a left common iliac arteriotomy and further randomized to a 1 × 3–cm patch angioplasty with either SIS or PTFE. All received cefotetan for 24 hours. All animals were sacrificed between 2 and 4 weeks, and necropsy was performed. Grafts were cultured, and microscopic analysis with hematoxylin and eosin and trichrome was performed. Outcomes included pulse quality (normal or diminished) compared with opposite side, graft infection, and pseudoaneurysm; all were determined by a blinded investigator. Results:Forty animals were randomized, and 1 died of abdominal sepsis. All control animals had normal distal pulses, no pseudoaneurysms, and no patch infections. The pseudoaneurysm rate for the contaminated PTFE patches was 25% compared with 0% in the SIS group (P = 0.09). Patch infection occurred in 73% of all PTFE patches compared with 8% of SIS patches (P < 0.03). Organisms present in the infected grafts included Escherichia coli, Bacteroides species, and other Gram-negative enterics. Histopathology demonstrated the presence of neointima in both SIS and PTFE. Only SIS was completely incorporated, with infiltration of collagen fibrils and lymphocytes. Conclusions:SIS was associated with improved graft patency, less infection, complete incorporation, and no false aneurysm formation when compared with PTFE. This may be due to its ability to provide a durable scaffold for cellularization and tissue remodeling. This material may offer a superior alternative to more complex vascular reconstruction techniques in contaminated fields.

MicroRNA-21 Promotes Glioblastoma Tumorigenesis by Down-regulating Insulin-like Growth Factor-binding Protein-3 (IGFBP3)

Background: miR-21 is overexpressed in many human cancers, including glioblastoma. Results: Insulin-like growth factor (IGF)-binding protein-3 (IGFBP3) is a novel miR-21 target gene and inhibits gliomagenesis in vitro and in vivo. Conclusion: miR-21 down-regulates IGFBP3, which acts as a tumor suppressor in human glioblastoma. Significance: IGFBP3 may have promise as a therapeutic target and prognostic marker for glioblastoma. Despite advances in surgery, imaging, chemotherapy, and radiation, patients with glioblastoma multiforme (GBM), the most common histological subtype of glioma, have an especially dismal prognosis; >70% of GBM patients die within 2 years of diagnosis. In many human cancers, the microRNA miR-21 is overexpressed, and accumulating evidence indicates that it functions as an oncogene. Here, we report that miR-21 is overexpressed in human GBM cell lines and tumor tissue. Moreover, miR-21 expression in GBM patient samples is inversely correlated with patient survival. Knockdown of miR-21 in GBM cells inhibited cell proliferation in vitro and markedly inhibited tumor formation in vivo. A number of known miR-21 targets have been identified previously. By microarray analysis, we identified and validated insulin-like growth factor (IGF)-binding protein-3 (IGFBP3) as a novel miR-21 target gene. Overexpression of IGFBP3 in glioma cells inhibited cell proliferation in vitro and inhibited tumor formation of glioma xenografts in vivo. The critical role that IGFBP3 plays in miR-21-mediated actions was demonstrated by a rescue experiment, in which IGFBP3 knockdown in miR-21KD glioblastoma cells restored tumorigenesis. Examination of tumors from GBM patients showed that there was an inverse relationship between IGFBP3 and miR-21 expression and that increased IGFBP3 expression correlated with better patient survival. Our results identify IGFBP3 as a novel miR-21 target gene in glioblastoma and suggest that the oncogenic miRNA miR-21 down-regulates the expression of IGFBP3, which acts as a tumor suppressor in human glioblastoma.

A multicenter study directly comparing the diagnostic accuracy of gene expression profiling and immunohistochemistry for primary site identification in metastatic tumors.

Metastatic tumors with an uncertain primary site can be a difficult clinical problem. In tens of thousands of patients every year, no confident diagnosis is ever issued, making standard-of-care treatment impossible. Gene expression profiling (GEP) tests currently available to analyze these difficult-to-diagnose tumors have never been directly compared with the diagnostic standard of care, immunochemistry (IHC). This prospectively conducted, blinded, multicenter study compares the diagnostic accuracy of GEP with IHC in identifying the primary site of 157 formalin-fixed paraffin-embedded specimens from metastatic tumors with known primaries, representing the 15 tissues on the GEP test panel. Four pathologists rendered diagnoses by selecting from 84 stains in 2 rounds. GEP was performed using the Pathwork Tissue of Origin Test. Overall, GEP accurately identified 89% of specimens, compared with 83% accuracy using IHC (P=0.013). In the subset of 33 poorly differentiated and undifferentiated carcinomas, GEP accuracy exceeded that of IHC (91% to 71%, P=0.023). In specimens for which pathologists rendered their final diagnosis with a single round of stains, both IHC and GEP exceeded 90% accuracy. However, when the diagnosis required a second round, IHC significantly underperformed GEP (67% to 83%, P<0.001). GEP has been validated as accurate in diagnosing the primary site in metastatic tumors. The Pathwork Tissue of Origin Test used in this study was significantly more accurate than IHC when used to identify the primary site, with the most pronounced superiority observed in specimens that required a second round of stains and in poorly differentiated and undifferentiated metastatic carcinomas.

Lymph node counts in uterine cancer: A randomized double blind trial

OBJECTIVE Higher number of lymph nodes counts may suggest a more accurate cancer staging. We wish to study whether sending lymph nodes to pathologist in four containers, instead of a single container, yields a higher nodal count. METHODS Patients with uterine cancer who underwent abdominal hysterectomy and lymphadenectomy were recruited. The right and left pelvic lymph nodes were collected from four locations (common, external and internal iliac, and obturator). Blinded randomization ex vivo allocated the side of the pelvic nodes specimen which was sent to pathology as one versus four containers. Each patient served as her own control by having the other side of her pelvic nodes sent as four different specimens. The surgeons and pathologists were blinded. RESULTS 104 consecutive patients were enrolled. The average age was 61 years old. The patients were predominately Caucasians (69%). The average total pelvic and aortic nodes per patients was 17.8. 54 patients, whose right-sided pelvic nodes were randomized to be sent in a single container, yielded an average of 7.2 right pelvic nodes versus 8.6 left pelvic nodes (p=0.026). 50 patients, whose left-sided pelvic nodes were randomized to be sent in a single container, yielded an average 8.1 right pelvic nodes versus 6.9 left pelvic nodes (p=0.042). CONCLUSION The lymph nodes count are higher when surgical nodes were sent as multiple separated instead of single specimens, regardless of the side of the pelvis.

VHL deletion impairs mammary alveologenesis but is not sufficient for mammary tumorigenesis.

Overexpression of hypoxia inducible factor-1 (HIF-1)alpha, which is common in most solid tumors, correlates with poor prognosis and high metastatic risk in breast cancer patients. Because HIF-1alpha protein stability is tightly controlled by the tumor suppressor von Hippel-Lindau (VHL), deletion of VHL results in constitutive HIF-1alpha expression. To determine whether VHL plays a role in normal mammary gland development, and if HIF-1alpha overexpression is sufficient to initiate breast cancer, Vhl was conditionally deleted in the mammary epithelium using the Cre/loxP system. During first pregnancy, loss of Vhl resulted in decreased mammary epithelial cell proliferation and impaired alveolar differentiation; despite these phenotypes, lactation was sufficient to support pup growth. In contrast, in multiparous dams, Vhl(-/-) mammary glands exhibited a progressive loss of alveolar epithelium, culminating in lactation failure. Deletion of Vhl in the epithelium also impacted the mammary stroma, as there was increased microvessel density accompanied by hemorrhage and increased immune cell infiltration. However, deletion of Vhl was not sufficient to induce mammary tumorigenesis in dams bred continuously for up to 24 months of age. Moreover, co-deletion of Hif1a could not rescue the Vhl(-/-)-dependent phenotype as dams were unable to successfully lactate during the first lactation. These results suggest that additional VHL-regulated genes besides HIF1A function to maintain the proliferative and regenerative potential of the breast epithelium.

Comparison of histopathology to gene expression profiling for the diagnosis of metastatic cancer

BackgroundDetermining the primary site of metastatic cancer with confidence can be challenging. Pathologists commonly use a battery of immunohistochemical (IHC) stains to determine the primary site. Gene expression profiling (GEP) has found increasing use, particularly in the most difficult cases. In this pilot study, a direct comparison between GEP and IHC-guided methods was performed.MethodsTen archived formalin-fixed paraffin embedded metastatic tumor samples for which the primary site had been clinically determined were selected. Five pathologists who were blinded to the diagnosis were asked to determine the primary site using IHC and other stains selected from a panel of 84 stains. Each pathologist was provided patient sex, biopsy site and gross sample description only. Slides were digitized using ScanScope®XT at 0.25 μm/pixel. Each evaluating pathologist was allowed to provide a diagnosis in three stages: initial (after reviewing the H&E image), intermediate (after reviewing images from the first batch of stains) and final diagnosis (after the second batch of stains if requested). GEP was performed using the only FDA-cleared test for this intended use, the Pathwork Tissue of Origin Test. No sample information was provided for GEP testing except for patient sex. Results were reported as the tumor tissue type with the highest similarity score.ResultsIn this feasibility study, GEP determined the correct primary site in 9 of the 10 cases (90%), compared to the IHC-guided method which determined the correct primary site for 32 of 50 case evaluations (average 64%, range 50% to 80%). The five pathologists directing the IHC-guided method ordered an average of 8.8 stains per case (range 1 to 18). GEP required an average of 3 slides per case (range 1 to 4).ConclusionsResults of the pilot study suggest that GEP provides correct primary site identification in a higher percentage of metastatic cases than IHC-guided methods, and uses less tissue. A larger comparative effectiveness study using this study design is needed to confirm the results.Virtual slidesThe virtual slide(s) for this article can be found here:http://www.diagnosticpathology.diagnomx.eu/vs/1749854104745508

An interferon response gene signature is associated with the therapeutic response of hepatitis C patients

Infection with the hepatitis C virus (HCV) is a major cause of chronic liver diseases and hepatocellular carcinoma worldwide, and thus represents a significant public health problem. The type I interferon (IFN), IFNα, has been successful in treating HCV-infected patients, but current IFN-based treatment regimens for HCV have suboptimal efficacy, and relatively little is known about why IFN therapy eliminates the virus in some patients but not in others. Therefore, it is critical to understand the basic mechanisms that underlie the therapeutic resistance to IFN action in HCV-infected individuals, and there is an urgent need to identify those patients most likely to respond to IFN therapy for HCV. To characterize the response of HCV-infected patients to treatment with IFNα, the expression of an IFN-response gene signature comprised of IFN-stimulated genes and genes that play an important role in the innate immune response was examined in liver biopsies from HCV-infected patients enrolled in a clinical trial. In the present study we found that the expression of a subset of IFN-response genes was dysregulated in liver biopsy samples from nonresponsive hepatitis C patients as compared with virologic responders. Based on these findings, a statistical model was developed to help predict the response of patients to IFN therapy, and compared to results obtained to the IL28 mutation model, which is highly predictive of the response to IFN-based therapy in HCV-infected patients. We found that a model incorporating gene expression data can improve predictions of IFN responsiveness compared to IL28 mutation status alone.

Absorbable biomaterials used within paranasal sinuses: an animal study.

Background The purpose of this study was to investigate the quality of mucosal regeneration in the presence of two different resorbable dressings derived from hyaluronic acid (HA): HA-carboxymethyl cellulose (HACMC) and esterified HA (HYAFF). A prospective randomized animal study was performed. Methods Twelve New Zealand white rabbits underwent bilateral maxillary sinusomy via a canine fossa approach. Each sinus was stripped circumferentially, except for the mucosa along the medial wall in the region of the natural ostium. Each of the 24 sinuses was then packed with either HACMC (n = 8) or HYAFF (n = 8) or left as an unpacked control (n = 8). After 14 days, each animal was killed and the sinus contents were evaluated histologically by a blinded pathologist. Results Criteria for optimal mucosal regeneration included a continuous layer of ciliated columnar epithelium with normal-appearing submucous glands and lack of both inflammatory infiltrate and fibrosis. Optimal regeneration was observed in 5/8 (62.5%) of the HACMC specimens, 1/8 (12.5%) of the HYAFF specimens, and 6/8 (75%) of the controls. The trend toward optimal regeneration using either HACMC or control was statistically significant when compared with HYAFF (p = 0.03). HYAFF specimens also were more likely to exhibit atrophic subepithelial glands in the regenerated mucosa. Polarizable foreign material was observed in 1/8 (12.5%) of the HACMC specimens and 2/8 (25%) of the HYAFF specimens. Conclusion The quality of epithelial regeneration is potentially affected by the form of HA present in the healing milieu. In this series, the most optimal healing characteristics were seen in unpacked controls. Between the preparations of HA studied, HACMC exhibited more favorable healing patterns, which were nearly similar to controls.