FGFR3 expression in primary invasive bladder cancers and matched lymph node metastases

PURPOSE FGFR3 is considered a good therapeutic target for bladder cancer. However, to our knowledge it is unknown whether the FGFR3 status of primary tumors is a surrogate for related metastases, which must be targeted by FGFR targeted systemic therapies. We assessed FGFR3 protein expression in primary bladder tumors and matched nodal metastases. MATERIALS AND METHODS We examined matched primary tumor and nodal metastases from 150 patients with bladder cancer clinically staged as N0M0. Four samples per patient were incorporated into a tissue microarray and FGFR3 expression was assessed by immunohistochemistry. FGFR3 expression was tested for an association with categorical clinical data using the Fisher exact test, and with overall and recurrence-free survival by Kaplan-Meier analysis. RESULTS Duplicate spots from primary tumors and lymph node metastases were highly concordant (OR 8.6 and 16.7, respectively, each p <0.001). Overall FGFR protein expression levels did not differ between primary and metastatic lesions (p = 0.78). Up-regulated expression was recorded in 53 of 106 evaluable primary tumor spots and 56 matched metastases. Concordance of FGFR3 expression levels in 79 matched primary tumor and metastasis specimens was high (OR 8.45, p <0.001). In 15 and 12 patients expression was up-regulated in only metastasis and in only the primary tumor, respectively. Overall and recurrence-free survival was not related to FGFR3 expression. CONCLUSIONS FGFR3 expression in matched primary and metastasized bladder cancer specimens showed good but not absolute concordance. Thus, in most patients primary tumor FGFR3 status can guide the selection of FGFR targeted therapy.

Bone marrow-derived mesenchymal stromal cells improve vascular regeneration and reduce leukocyte-endothelium activation in critical ischemic murine skin in a dose-dependent manner

BACKGROUND AIMS Stem cells participate in vascular regeneration following critical ischemia. However, their angiogenic and remodeling properties, as well as their role in ischemia-related endothelial leukocyte activation, need to be further elucidated. Herein, we investigated the effect of bone marrow-derived mesenchymal stromal cells (BM-MSCs) in a critically ischemic murine skin flap model. METHODS Groups received either 1 × 10(5), 5 × 10(5), or 1 × 10(6) BM-MSCs or cell-free conditioned medium (CM). Controls received sodium chloride. Intravital fluorescence microscopy was performed for morphological and quantitative assessment of micro-hemodynamic parameters over 12 days. RESULTS Tortuosity and diameter of conduit-arterioles were pronounced in the MSC groups (P < 0.01), whereas vasodilation was shifted to the end arteriolar level in the CM group (P < 0.01). These effects were accompanied by angiopoietin-2 expression. Functional capillary density and red blood cell velocity were enhanced in all treatment groups (P < 0.01). Although a significant reduction of rolling and sticking leukocytes was observed in the MSC groups with a reduction of diameter in postcapillary venules (P < 0.01), animals receiving CM exhibited a leukocyte-endothelium interaction similar to controls. This correlated with leukocyte common antigen expression in tissue sections (P < 0.01) and p38 mitogen-activated protein kinase expression from tissue samples. Cytokine analysis from BM-MSC culture medium revealed a 50% reduction of pro-inflammatory cytokines (interleukin [IL]-1β, IL-6, IL-12, tumor necrosis factor-α, interferon-γ) and chemokines (keratinocyte chemoattractant, granulocyte colony-stimulating factor) under hypoxic conditions. DISCUSSION We demonstrated positive effects of BM-MSCs on vascular regeneration and modulation of endothelial leukocyte adhesion in critical ischemic skin. The improvements after MSC application were dose-dependent and superior to the use of CM alone.

Long-term results of a prospective randomised trial assessing the impact of readaptation of the dorsolateral peritoneal layer following extended pelvic lymph node dissection and cystectomy.

OBJECTIVE To evaluate the long term oncological and functional outcomes after readaptation of the dorsolateral peritoneal layer following pelvic lymph node dissection (PLND) and cystectomy . PATIENTS AND METHODS A randomised, single-center, single-blinded, two-arm trial was conducted on 200 consecutive cystectomy patients who underwent PLND and cystectomy for bladder cancer (

Implications of intraglandular lymph node metastases in primary carcinomas of the parotid gland

OBJECTIVES/HYPOTHESIS Assess the diagnostic and prognostic relevance of intraglandular lymph node (IGLN) metastases in primary parotid gland carcinomas (PGCs). STUDY DESIGN Retrospective study at a tertiary referral university hospital. METHODS We reviewed the records of 95 patients with primary PGCs, treated at least surgically, between 1997 and 2010. We assessed the clinicopathological associations of IGLN metastases, their prognostic significance, and predictive value in the diagnosis of occult neck lymph node metastases RESULTS Twenty-four (25.26%) patients had IGLN metastases. This feature was significantly more prevalent in patients with advanced pT status (P = .01), pN status (P < .01), and overall stage (P < .001); high-risk carcinomas (P = .01); as well as in patients with treatment failures (P < .01). IGLN involvement was significantly associated with decreased univariate disease-free survival (P < .001). Positive and negative predictive values and accuracy for IGLN involvement in the detection of occult neck lymph node metastases were 63.64%, 90.48%, and 84.91%, respectively. The diagnostic values were generally higher in patients with low-risk subtype of PGCs. CONCLUSIONS IGLN involvement provides prognostic information and is associated with advanced tumoral stage and higher risk of recurrence. This feature could be used as a potential readout to determine whether a neck dissection in clinically negative neck lymph nodes is needed or not. LEVEL OF EVIDENCE 4.

Osteogenic differentiation of bone marrow stromal cells is hindered by the presence of intervertebral disc cells.

BACKGROUND Clinical observations indicate that the presence of nucleus pulposus (NP) tissue during spinal fusion hinders the rate of disc ossification. While the underlying mechanism remains unknown, this observation could be due to incomplete removal of NP cells (NPCs) that secrete factors preventing disc calcification, such as bone morphogenetic protein (BMP) antagonists including noggin and members of the DAN (differential screening selected gene aberrative in neuroblastoma) family. METHODS Monolayer human bone marrow-derived mesenchymal stem cells (MSCs) were cocultured withNPCs and annulus fibrosus cells (AFCs) embedded in alginate for 21 days. At the end of coculture, MSCs were stained for mineral deposition by alizarin red, and relative expression of bone-related genes [Runt-related transcription factor 2, (RUNX2), Osteopontin (OPN), and Alkaline phosphatase (ALP)] and ALP activity were analyzed. Relative expression of three BMP antagonists, chordin (CHRD), gremlin (GREM1), and noggin (NOG), was determined in primary human NPCs and AFCs. These cells were also stained for Gremlin and Noggin by immunocytochemistry. RESULTS Alizarin red staining showed that MSC osteogenesis in monolayer cultures was inhibited by coculture with NPCs or AFCs. ALP activity and RT-PCR analyses confirmed these results and demonstrated inhibition of osteogenesis of MSC in the presence of disc cells. NOG was significantly up-regulated in MSCs after coculture. Relative gene expression of intervertebral disc (IVD) cells showed higher expression of GREM1 in NPCs than in AFCs. CONCLUSIONS We show that primary IVD cells inhibit osteogenesis of MSCs. BMP inhibitors NOG, GREM1 and CHRD were expressed in IVD cells. GREM1 appears to be differentially expressed in NPCs and AFCs. Our results have implications for the design and development of treatments for non-union in spinal fusion.

Sentinel lymph node biopsy in thick malignant melanoma: A 16-year single unit experience.

BACKGROUND The role of sentinel lymph node biopsy (SLNB) and its benefits in patients with thick melanoma is still controversial. OBJECTIVES We evaluated the clinical effect of SLNB in patients with thick melanoma. METHODS We performed a retrospective cohort review (1996-2012) of thick melanomas. Collected data included the patient and tumour characteristics. Locoregional recurrence, distant metastases, disease free and overall survival were compared between the patients with positive and negative SLNB. RESULTS 126 thick melanomas with a mean age of 64.09 years were included in the study. Positive SLNB were found in 47 (37.3%) patients. Significantly more locoregional recurrence (P = 0.002) and distant metastases (P = 0.030) were detected in the patients with positive SLNB. Furthermore, the patients with negative SLNB showed significantly better disease free survival (P = 0.021). CONCLUSIONS Positive SLNB might be prognostic factor in thick melanoma and aggravates the outcome of thick melanomas.

Human graft-derived mesenchymal stromal cells potently suppress alloreactive T-cell responses

After organ transplantation, recipient T cells contribute to graft rejection. Mesenchymal stromal cells from the bone marrow (BM-MSCs) are known to suppress allogeneic T-cell responses, suggesting a possible clinical application of MSCs in organ transplantation. Human liver grafts harbor resident populations of MSCs (L-MSCs). We aimed to determine the immunosuppressive effects of these graft-derived MSCs on allogeneic T-cell responses and to compare these with the effects of BM-MSCs. BM-MSCs were harvested from aspirates and L-MSCs from liver graft perfusates. We cultured them for 21 days and compared their suppressive effects with the effects of BM-MSCs on allogeneic T-cell responses. Proliferation, cytotoxic degranulation, and interferon-gamma production of alloreactive T cells were more potently suppressed by L-MSCs than BM-MSCs. Suppression was mediated by both cell-cell contact and secreted factors. In addition, L-MSCs showed ex vivo a higher expression of PD-L1 than BM-MSCs, which was associated with inhibition of T-cell proliferation and cytotoxic degranulation in vitro. Blocking PD-L1 partly abrogated the inhibition of cytotoxic degranulation by L-MSCs. In addition, blocking indoleamine 2,3-dioxygenase partly abrogated the inhibitive effects of L-MSCs, but not BM-MSCs, on T-cell proliferation. In conclusion, liver graft-derived MSC suppression of allogeneic T-cell responses is stronger than BM-MSCs, which may be related to in situ priming and mobilization from the graft. These graft-derived MSCs may therefore be relevant in transplantation by promoting allohyporesponsiveness.

Laparoscopic Indocyanine Green Sentinel Lymph Node Mapping in Pregnant Cervical Cancer Patients.

We present cases of 2 pregnant patients with early-stage cervical cancer who have undergone indocyanine green (ICG) sentinel lymph node (SLN) mapping followed by laparoscopic SLN biopsy, pelvic lymphadenectomy, and cervical conization. Eight milliliters of ICG were injected in the 4 quadrants of the cervix after having obtained an adequate pneumoperitoneum and having inspected the abdominal cavity. SLNs were identified in both hemipelvises in both patients. In the final pathologic analysis, both SLNs and non-SLNs were negative for metastatic disease. No adverse events from ICG injection were recorded. ICG SLN mapping seems to be feasible in pregnant cervical cancer patients.

A Comparison of Radiocolloid and Indocyanine Green Fluorescence Imaging, Sentinel Lymph Node Mapping in Patients with Cervical Cancer Undergoing Laparoscopic Surgery.

BACKGROUND AND PURPOSE (99)TC combined with blue-dye mapping is considered the best sentinel lymph node (SLN) mapping technique in cervical cancer. Indocyanine green (ICG) with near infrared fluorescence imaging has been introduced as a new methodology for SLN mapping. The aim of this study was to compare these two techniques in the laparoscopic treatment of cervical cancer. METHODS Medical records of patients undergoing laparoscopic SLN mapping for cervical cancer with either (99)Tc and patent blue dye (Group 1) or ICG (Group 2) from April 2008 until August 2012 were reviewed. Sensitivity, specificity, and overall and bilateral detection rates were calculated and compared. RESULTS Fifty-eight patients were included in the study-36 patients in Group 1 and 22 patients in Group 2. Median tumor diameter was 25 and 29 mm, and mean SLN count was 2.1 and 3.7, for Groups 1 and 2, respectively. Mean non-SLN (NSLN) count was 39 for both groups. SLNs were ninefold more likely to be affected by metastatic disease compared with NSLNs (p < 0.005). Sensitivity and specificity were both 100 %. Overall detection rates were 83 and 95.5 % (p = nonsignificant), and bilateral detection rates were 61 and 95.5 % (p < 0.005), for Groups 1 and 2, respectively. In 75 % of cases, SLNs were located along the external or internal iliac nodal basins. CONCLUSIONS ICG SLN mapping in cervical cancer provides high overall and bilateral detection rates that compare favorably with the current standard of care.

A Specific Mapping Study Using Fluorescence Sentinel Lymph Node Detection in Patients with Intermediate- and High-risk Prostate Cancer Undergoing Extended Pelvic Lymph Node Dissection.

Sentinel lymph node (SLN) detection techniques have the potential to change the standard of surgical care for patients with prostate cancer. We performed a lymphatic mapping study and determined the value of fluorescence SLN detection with indocyanine green (ICG) for the detection of lymph node metastases in intermediate- and high-risk patients undergoing radical prostatectomy and extended pelvic lymph node dissection. A total of 42 patients received systematic or specific ICG injections into the prostate base, the midportion, the apex, the left lobe, or the right lobe. We found (1) that external and internal iliac regions encompass the majority of SLNs, (2) that common iliac regions contain up to 22% of all SLNs, (3) that a prostatic lobe can drain into the contralateral group of pelvic lymph nodes, and (4) that the fossa of Marcille also receives significant drainage. Among the 12 patients who received systematic ICG injections, 5 (42%) had a total of 29 lymph node metastases. Of these, 16 nodes were ICG positive, yielding 55% sensitivity. The complex drainage pattern of the prostate and the low sensitivity of ICG for the detection of lymph node metastases reported in our study highlight the difficulties related to the implementation of SNL techniques in prostate cancer. PATIENT SUMMARY There is controversy about how extensive lymph node dissection (LND) should be during prostatectomy. We investigated the lymphatic drainage of the prostate and whether sentinel node fluorescence techniques would be useful to detect node metastases. We found that the drainage pattern is complex and that the sentinel node technique is not able to replace extended pelvic LND.

The role of SRC family kinase activation in prostate cancer growth and lymph node metastasis

Aberrant expression and/or activation of Src Family of non-receptor protein tyrosine kinases (SFKs) occur frequently during progressive stages of multiple types of human malignancies, including prostate cancer. Two SFKs, Src and Lyn, are expressed and implicated in prostate cancer progression. Work in this dissertation investigated the specific roles of Src and Lyn in the prostate tumor progression, and the effects of SFK inhibition on prostate tumor growth and lymph node metastasis in pre-clinical mouse models. ^ Firstly, using a pharmacological inhibitor of SFKs in clinical trials, dasatinib, I demonstrated that SFK inhibition affects both cellular migration and proliferation in vitro. Systemic administration of dasatinib reduced primary tumor growth, as well as development of lymph node metastases, in both androgen-sensitive and -resistant orthotopic prostate cancer mouse models. Immunohistochemical analysis of the primary tumors revealed that dasatinib treatment decreased SFK phosphorylation but not expression, resulting in decreased cellular proliferation and increased apoptosis. For this analysis of immunohistochemical stained tissues, I developed a novel method of quantifying immunohistochemical stain intensity that greatly reduced the inherent bias in analyzing staining intensity. ^ To determine if Src and Lyn played overlapping or distinct roles in prostate cancer tumor growth and progression, Src expression alone was inhibited by small-interfering RNA. The resulting stable cell lines were decreased in migration, but not substantially affected in proliferation rates. In contrast, an analogous strategy targeting Lyn led to stable cell lines in which proliferation rates were significantly reduced. ^ Lastly, I tested the efficacy of a novel SFK inhibitor (KX2-391) targeting peptide substrate-binding domain, on prostate cancer growth and lymph node metastasis in vivo. I demonstrated that KX2-391 has similar effects as dasatinib, an ATP-competitive small molecular inhibitor, on both the primary tumor growth and development of lymph node metastasis in vivo, work that contributed to the first-in-man Phase I clinical trial of KX2-391. ^ In summary, studies in this dissertation provide the first demonstration that Src and Lyn activities affect different cellular functions required for prostate tumor growth and metastasis, and SFK inhibitors effectively reduce primary tumor growth and lymph node metastasis. Therefore, I conclude that SFKs are promising therapeutic targets for treatment of human prostate cancer. ^

IDENTIFICATION AND CHARACTERIZATION OF DISTINCT POPULATIONS OF CLONOGENIC BONE MARROW STROMAL CELLS CAPABLE OF TRANSFERRING THE HEMATOPOIETIC MICROENVIRONMENT IN VIVO AND SUPPORTING LT-HSCs IN VITRO

Bone marrow (BM) stromal cells are ascribed two key functions, 1) stem cells for non-hematopoietic tissues (MSC) and 2) as components of the hematopoietic stem cell niche. Current approaches studying the stromal cell system in the mouse are complicated by the low yield of clonogenic progenitors (CFU-F). Given the perivascular location of MSC in BM, we developed an alternative methodology to isolate MSC from mBM. An intact ‘plug’ of bone marrow is expelled from bones and enzymatically disaggregated to yield a single cell suspension. The recovery of CFU-F (1917.95+199) reproducibly exceeds that obtained using the standard BM flushing technique (14.32+1.9) by at least 2 orders of magnitude (P<0.001; N = 8) with an accompanying 196-fold enrichment of CFU-F frequency. Purified BM stromal and vascular endothelial cell populations are readily obtained by FACS. A detailed immunophenotypic analysis of lineage depleted BM identified PDGFRαβPOS stromal cell subpopulations distinguished by their expression of CD105. Both subpopulations retained their original phenotype of CD105 expression in culture and demonstrate MSC properties of multi-lineage differentiation and the ability to transfer the hematopoietic microenvironment in vivo. To determine the capacity of either subpopulation to support long-term multi-lineage reconstituting HSCs, we fractionated BM stromal cells into either the LinNEGPDGFRαβPOSCD105POS and LINNEGPDGFRαβPOSCD105LOW/- populations and tested their capacity to support LT-HSC by co-culturing each population with either 1 or 10 HSCs for 10 days. Following the 10 day co-culture period, both populations supported transplantable HSCs from 10 cells/well co-cultures demonstrating high levels of donor repopulation with an average of 65+23.6% chimerism from CD105POS co-cultures and 49.3+19.5% chimerism from the CD105NEG co-cultures. However, we observed a significant difference when mice were transplanted with the progeny of a single co-cultured HSC. In these experiments, CD105POS co-cultures (100%) demonstrated long-term multi- lineage reconstitution, while only 4 of 8 mice (50%) from CD105NEG -single HSC co-cultures demonstrated long-term reconstitution, suggesting a more limited expansion of functional stem cells. Taken together, these results demonstrate that the PDGFRαβCD105POS stromal cell subpopulation is distinguished by a unique capacity to support the expansion of long-term reconstituting HSCs in vitro.

Connexin-43 prevents hematopoietic stem cell senescence through transfer of reactive oxygen species to bone marrow stromal cells

Hematopoietic stem cell (HSC) aging has become a concern in chemotherapy of older patients. Humoral and paracrine signals from the bone marrow (BM) hematopoietic microenvironment (HM) control HSC activity during regenerative hematopoiesis. Connexin-43 (Cx43), a connexin constituent of gap junctions (GJs) is expressed in HSCs, down-regulated during differentiation, and postulated to be a self-renewal gene. Our studies, however, reveal that hematopoietic-specific Cx43 deficiency does not result in significant long-term competitive repopulation deficiency. Instead, hematopoietic Cx43 (H-Cx43) deficiency delays hematopoietic recovery after myeloablation with 5-fluorouracil (5-FU). 5-FU-treated H-Cx43-deficient HSC and progenitors (HSC/P) cells display decreased survival and fail to enter the cell cycle to proliferate. Cell cycle quiescence is associated with down-regulation of cyclin D1, up-regulation of the cyclin-dependent kinase inhibitors, p21cip1. and p16INK4a, and Forkhead transcriptional factor 1 (Foxo1), and activation of p38 mitogen-activated protein kinase (MAPK), indicating that H-Cx43-deficient HSCs are prone to senescence. The mechanism of increased senescence in H-Cx43-deficient HSC/P cells depends on their inability to transfer reactive oxygen species (ROS) to the HM, leading to accumulation of ROS within HSCs. In vivo antioxidant administration prevents the defective hematopoietic regeneration, as well as exogenous expression of Cx43 in HSC/P cells. Furthermore, ROS transfer from HSC/P cells to BM stromal cells is also rescued by reexpression of Cx43 in HSC/P. Finally, the deficiency of Cx43 in the HM phenocopies the hematopoietic defect in vivo. These results indicate that Cx43 exerts a protective role and regulates the HSC/P ROS content through ROS transfer to the HM, resulting in HSC protection during stress hematopoietic regeneration.

Unique molecular surface features of in vivo tolerized T cells

Differential expression of surface markers can frequently be used to distinguish functional subsets of T cells, yet a surface phenotype unique to T cells induced into an anergic state has not been described. Here, we report that CD4 T cells rendered anergic in vivo by superantigen can be identified by loss of the 6C10 T cell marker. Inoculation of Vβ8.1 T cell antigen receptor (TCR) transgenic mice with a Vβ8.1-reactive minor lymphocyte-stimulating superantigen (Mls-1a) induces tolerance to Mls-1a by clonal anergy. CD4 lymph node T cells from Mls-1a inoculated transgenic mice enriched for the 6C10− phenotype neither proliferate nor produce interleukin-2 upon TCR engagement, whereas 6C10+ CD4 T cells retain responsiveness. Analysis of T cell memory markers demonstrate that 6C10− T cells remain 3G11hi but express heterogeneous levels of CD45RB, CD62L, CD44, and the CD69 early activation marker, suggesting that T cells at various degrees of activation can be functionally anergic. These studies demonstrate that anergic T cells can be purified based on 6C10 expression permitting examination of issues concerning biochemical and biological features specific to T cell anergy.