Judith Sandbank

G-CSF induces stem cell mobilization by decreasing bone marrow SDF-1 and up-regulating CXCR4

Granulocyte colony-stimulating factor (G-CSF)–induced hematopoietic stem cell mobilization is widely used for clinical transplantation; however, the mechanism is poorly understood. We report here that G-CSF induced a reduction of the chemokine stromal cell–derived factor 1 (SDF-1) and an increase in its receptor CXCR4 in the bone marrow (BM), whereas their protein expression in the blood was less affected. The gradual decrease of BM SDF-1, due mostly to its degradation by neutrophil elastase, correlated with stem cell mobilization. Elastase inhibition reduced both activities. Human and murine stem cell mobilization was inhibited by neutralizing CXCR4 or SDF-1 antibodies, demonstrating SDF-1–CXCR4 signaling in cell egress. We suggest that manipulation of SDF-1–CXCR4 interactions may be a means with which to control the navigation of progenitors between the BM and blood to improve the outcome of clinical stem cell transplantation.

Induction of the chemokine stromal-derived factor-1 following DNA damage improves human stem cell function

The chemokine stromal-derived factor-1 (SDF-1) controls many aspects of stem cell function. Details of its regulation and sites of production are currently unknown. We report that in the bone marrow, SDF-1 is produced mainly by immature osteoblasts and endothelial cells. Conditioning with DNA-damaging agents (ionizing irradiation, cyclophosphamide, and 5-fluorouracil) caused an increase in SDF-1 expression and in CXCR4-dependent homing and repopulation by human stem cells transplanted into NOD/SCID mice. Our findings suggest that immature osteoblasts and endothelial cells control stem cell homing, retention, and repopulation by secreting SDF-1, which also participates in host defense responses to DNA damage.

The chemokine SDF-1 stimulates integrin-mediated arrest of CD34+ cells on vascular endothelium under shear flow

The chemokine SDF-1 plays a central role in the repopulation of the bone marrow (BM) by circulating CD34(+) progenitors, but the mechanisms of its action remain obscure. To extravasate to target tissue, a blood-borne cell must arrest firmly on vascular endothelium. Murine hematopoietic progenitors were recently shown in vivo to roll along BM microvessels that display selectins and integrins. We now show that SDF-1 is constitutively expressed by human BM endothelium. In vitro, human CD34(+) cells establish efficient rolling on P-selectin, E-selectin, and the CD44 ligand hyaluronic acid under physiological shear flow. ICAM-1 alone did not tether CD34(+) cells under flow, but, in the presence of surface-bound SDF-1, CD34(+) progenitors rolling on endothelial selectin rapidly developed firm adhesion to the endothelial surface, mediated by an interaction between ICAM-1 and its integrin ligand, which coimmobilized with SDF-1. Human CD34(+) cells accumulated efficiently on TNF-activated human umbilical cord endothelial cells in the absence of SDF-1, but they required immobilized SDF-1 to develop firm integrin-mediated adhesion and spreading. In the absence of selectins, SDF-1 also promoted VLA-4-mediated, Gi protein-dependent tethering and firm adhesion to VCAM-1 under shear flow. To our knowledge, this is the first demonstration that SDF-1 expressed on vascular endothelium is crucial for translating rolling adhesion of CD34(+) progenitors into firm adhesion by increasing the adhesiveness of the integrins VLA-4 and LFA-1 to their respective endothelial ligands, VCAM-1 and ICAM-1.

Leydig Cell Tumors of the Testis Gray Scale and Color Doppler Sonographic Appearance

Objective. To determine the gray scale and color Doppler sonographic features of Leydig cell tumors of the testis in a series of patients. Methods. We retrospectively analyzed the sonographic appearance of 10 proven Leydig cell tumors in 9 patients aged 26 to 47 years. Sonographic features that were reviewed included the size and echogenicity of the tumors, presence of cystic areas or calcifications, and distribution pattern of detectable blood flow on color or power Doppler imaging. Results. The tumors ranged from 0.4 to 3.0 cm in diameter, but most were less than 1.0 cm in diameter. In 1 testis, 2 discrete Leydig cell tumors were found. Nine (90%) of the 10 tumors were homogeneously hypoechoic. Only 1 tumor was isoechoic with the testis. None of the tumors contained calcifications. Of 8 tumors with color Doppler imaging, 7 (88%) showed a characteristic pattern of increased peripheral blood flow, which was either circumferential or punctate. Only 1 tumor was found with internal hypervascularity. Conclusions. Peripheral hypervascularity in a hypoechoic testicular tumor that has little or no internal color Doppler flow should suggest the possibility of a Leydig cell tumor, and consideration should be given to testicle‐sparing surgery.

Angiogenesis in pterygium: Morphometric and immunohistochemical study

Objective. To evaluate the role of angiogenesis in the pathogenesis of pterygium by comparing the expression of von-Willebrand factor (vWF) and vascular endothelial growth factor (VEGF) in pterygium, and in normal superior bulbar conjunctiva. Methods. 23 human samples from pterygium and the superior bulbar conjunctiva were stained using rabbit anti-vWF and anti-VEGF antibodies. The density of vWF and VEGF positive vessels, VEGF staining intensity and the number of VEGF positive stromal, epithelial and vascular endothelial cells were evaluated. Results. Pterygium specimens had higher average vWF and VEGF positive microvascular counts per high power field (P = 0.0012), higher average VEGF staining intensity scores in epithelial, stromal and endothelial cells (p < 0.0001) and higher VEGF positive cell counts (P < 0.0001) than normal conjuctiva. Conclusions. Over-expression of VEGF in pterygium tissue, together with the abundance of vWF-stained new vessels, may support previous suggestions that angiogenesis may play a role in the formation of pterygium.

Potential role of intratumor bacteria in mediating tumor resistance to the chemotherapeutic drug gemcitabine

In model systems, bacteria present in human pancreatic tumors confer resistance to the anticancer drug gemcitabine. Debugging a cancer therapy Microbes contribute not only to the development of human diseases but also to the response of diseases to treatment. Geller et al. show that certain bacteria express enzymes capable of metabolizing the cancer chemotherapeutic drug gemcitabine into an inactive form. When bacteria were introduced into tumors growing in mice, the tumors became resistant to gemcitabine, an effect that was reversed by antibiotic treatment. Interestingly, a high percentage of human pancreatic ductal adenocarcinomas, a tumor type commonly treated with gemcitabine, contain the culprit bacteria. These correlative results raise the tantalizing possibility that the efficacy of an existing therapy for this lethal cancer might be improved by cotreatment with antibiotics. Science, this issue p. 1156 Growing evidence suggests that microbes can influence the efficacy of cancer therapies. By studying colon cancer models, we found that bacteria can metabolize the chemotherapeutic drug gemcitabine (2′,2′-difluorodeoxycytidine) into its inactive form, 2′,2′-difluorodeoxyuridine. Metabolism was dependent on the expression of a long isoform of the bacterial enzyme cytidine deaminase (CDDL), seen primarily in Gammaproteobacteria. In a colon cancer mouse model, gemcitabine resistance was induced by intratumor Gammaproteobacteria, dependent on bacterial CDDL expression, and abrogated by cotreatment with the antibiotic ciprofloxacin. Gemcitabine is commonly used to treat pancreatic ductal adenocarcinoma (PDAC), and we hypothesized that intratumor bacteria might contribute to drug resistance of these tumors. Consistent with this possibility, we found that of the 113 human PDACs that were tested, 86 (76%) were positive for bacteria, mainly Gammaproteobacteria.

Diagnostic Performance of a Novel Device for Real-Time Margin Assessment in Lumpectomy Specimens

BACKGROUND Margin status in breast lumpectomy procedures is a prognostic factor for local recurrence and the need to obtain clear margins is often a cause for repeated surgical procedures. A recently developed device for real-time intraoperative margin assessment (MarginProbe; Dune Medical Devices, Caesarea, Israel), was clinically tested. The work presented here looks at the diagnostic performance of the device. METHODS The device was applied to freshly excised lumpectomy and mastectomy specimens at specific tissue measurement sites. These measurement sites were accurately marked, cut out, and sent for histopathologic analysis. Device readings (positive or negative) were compared with histology findings (namely malignant, containing any microscopically detected tumor, or nonmalignant) on a per measurement site basis. The sensitivity and specificity of the device was computed for the full dataset and for additional relevant subgroups. RESULTS A total of 869 tissue measurement sites were obtained from 76 patients, 753 were analyzed, of which 165 were cancerous and 588 were nonmalignant. Device performance on relatively homogeneous sites was: sensitivity 1.00 (95% CI: 0.85-1), specificity 0.87 (95% CI: 0.83-0.90). Performance for the full dataset was: sensitivity 0.70 (95% CI: 0.63-0.77), specificity 0.70 (95% CI: 0.67-0.74). Device sensitivity was estimated to change from 56% to 97% as the cancer feature size increased from 0.7 mm to 6.6 mm. Detection rate of samples containing pure DCIS clusters was not different from rates of samples containing IDC. CONCLUSIONS The device has high sensitivity and specificity in distinguishing between normal and cancer tissue even down to small cancer features.

Positive surgical margins with renal cell carcinoma have a limited influence on long-term oncological outcomes of nephron sparing surgery.

OBJECTIVES To define the rate of positive surgical margins (PSMs) and analyze the outcome of patients with PSMs. The outcome and proper management of patients with positive PSMs during nephron sparing surgery (NSS) are questionable. In this study we define the clinical outcomes of PSMs at NSS and suggest management. METHODS Clinical records of 114 renal units who underwent open NSS for a renal mass between May 1995 and September 2005 were reviewed. RESULTS PSMs were suspected on frozen section in 17 of 114 renal units (15%). Tumors with suspected PSMs at frozen section were smaller (2.9 +/- 1.6) in comparison to those with negative surgical margins (3.4 +/- 1.8 cm) (P = .001). Nine of 17 (53%) cases underwent total nephrectomy (5 immediately, 4 delayed). In 4 (24%), immediate re-excision of the renal crater was performed. A total of 4 (24%) that were followed up clinically were with no evidence of disease. Therefore, in 13 of 17 (77%) cases, the presence of tumor cells at the remaining side of the kidney could be evaluated histologically. In 2 cases from the immediate response group, tumor cells were found in the side opposite to the resection. There was no residual tumor in any case subjected to delayed nephrectomy. At median follow-up of 71 months, 15 of 17 patients are alive and with no evidence of disease. Two patients died because of unrelated causes. The overall 5-year survival rate is 98.2% and there is no cancer-specific mortality. CONCLUSIONS The true PSM rate is in the range of 1.75%-5.26%. No disease progression or deaths attributable to renal cell carcinoma were associated with PSMs. Total nephrectomy should be avoided as a response to PSMs.

Serum DNA methylation for monitoring response to neoadjuvant chemotherapy in breast cancer patients

Patients with large or nonoperable breast cancers often receive neoadjuvant chemotherapy to facilitate full resection of the tumor and enable conservation of the breast. However, currently available methods for evaluation of response during therapy are limited and the actual effect of the treatment is only recognized at surgery upon completion of chemotherapy. Timely assessment of response could allow individual tailoring of the treatment and save noneffective drugs and unnecessary toxicity. Here, we suggest that tumor derived DNA methylation in the serum may reflect changes in tumor burden and allow early recognition of responders versus nonresponders. In this pilot study, we collected 7 consecutive serum samples from 52 patients with locally advanced breast cancer during neoadjuvant chemotherapy. We selected RASSF1, which was methylated in more than 80% of the tumors, for serum analysis. Using the “methylation sensitive PCR and high resolution melting,” we detected RASSF1 methylation in the serum of 21 patients prior to therapy. In four patients who achieved complete pathological response, RASSF1 methylation in the serum became undetectable early during therapy. In contrast, in 17 patients that had partial or minimal pathological response, serum RASSF1 methylation persisted longer or throughout the treatment (complete versus partial response p = 0.02). These findings support further development of this assay for monitoring response during neoadjuvant therapy.

Lymphoma and Leukemia Cells Possess Fractal Dimensions That Correlate with Their Biological Features

Background: Living cells can be viewed as complex adaptive systems that exhibit non-linear dynamics and fractal features. We investigated the fractal qualities of normal and malignant hematological cells and their potential as a tool for characterizing cell phenotype and clinical behavior. Methods: A mathematical algorithm and an optic tool for fractal analysis of nuclei were developed. A total of 4,713 lymphoid cells derived from 66 patients of five distinct diagnostic groups (normal and reactive lymphocytes, low-grade lymphomas and an aggressive lymphoma) were assessed for their fractal dimension. In addition, in 19 patients fractal analysis of leukemia cells was compared to clinical endpoints. Results: After validating our method, hematological cells possessed fractal dimensions corresponding to their clinical entity. There was a highly significant overall difference in fractal dimensions between various types of hematological malignancies. A preliminary correlation was found between the fractal dimension and the clinical outcome of leukemia patients. Conclusions: Hematological cells possess fractal dimensions that correlate with their biological properties. Measurement of fractal dimension seems to be a sensitive method to assess the hematological cell phenotype and to define a clinical group. This tool may be potentially useful for the evaluation of clinical behavior of hematological diseases.