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1.  Ex vivo localization and immunohistochemical detection of sentinel lymph node micrometastasis in patients with colorectal cancer can upgrade tumor staging 
Diagnostic Pathology  2012;7:71.
It is not clear if sentinel lymph node (SLN) mapping can improve outcomes in patients with colorectal cancers. The purpose of this study was to determine the prognostic values of ex vivo sentinel lymph node (SLN) mapping and immunohistochemical (IHC) detection of SLN micrometastasis in colorectal cancers.
Colorectal cancer specimens were obtained during radical resections and the SLN was identified by injecting a 1% isosulfan blue solution submucosally and circumferentially around the tumor within 30 min after surgery. The first node to stain blue was defined as the SLN. SLNs negative by hematoxylin and eosin (HE) staining were further examined for micrometastasis using cytokeratin IHC.
A total of 54 patients between 25 and 82 years of age were enrolled, including 32 males and 22 females. More than 70% of patients were T3 or above, about 86% of patients were stage II or III, and approximately 90% of patients had lesions grade II or above. Sentinel lymph nodes were detected in all 54 patients. There were 32 patients in whom no lymph node micrometastasis were detected by HE staining and 22 patients with positive lymph nodes micrometastasis detected by HE staining in non-SLNs. In contrast only 7 SLNs stained positive with HE. Using HE examination as the standard, the sensitivity, non-detection rate, and accuracy rate of SLN micrometastasis detection were 31.8% (7/22), 68.2% (15/22), and 72.2%, respectively. Micrometastasis were identified by ICH in 4 of the 32 patients with HE-negative stained lymph nodes, resulting in an upstaging rate 12.5% (4/32). The 4 patients who were upstaged consisted of 2 stage I patients and 2 stage II patients who were upstaged to stage III. Those without lymph node metastasis by HE staining who were upstaged by IHC detection of micrometastasis had a significantly poorer disease-free survival (p = 0.001) and overall survival (p = 0.004).
Ex vivo localization and immunohistochemical detection of sentinel lymph node micrometastasis in patients with colorectal cancer can upgrade tumor staging, and may become a factor affecting prognosis and guiding treatment.
Virtual slides
The virtual slide(s) for this article can be found here:
PMCID: PMC3472318  PMID: 22726450
Colorectal carcinoma; Sentinel lymph node; Micrometastasis; Prognosis
2.  Human High Molecular Weight–Melanoma-Associated Antigen: Utility for Detection of Metastatic Melanoma in Sentinel Lymph Nodes 
Detection of micrometastasis in melanoma-draining lymph nodes is important for staging and prognosis. Immunohistochemical staining (IHC) using S-100p-HMB-45–, and MART-1– specfic antibodies is used for detecting metastases in sentinel lymph nodes (SLN). However, improvement in IHC is needed for melanoma micrometastasis detection.
Experimental Design
Paraffin-embedded archival tissue (PEAT) specimens were obtained from 42 non-SLN macrometastases, 42 SLN metastases, and 16 tumor-negative SLNs of 100 melanoma patients who underwent SLN biopsy. PEAT specimens were assessed by IHC with high molecular weight-melanoma-associated antigen (HMW-MAA)– specific monoclonal antibodies (mAb) and with S-100p-, HMB-45–, and MART-1– specific antibodies. Quantitative real-time reverse-transcriptase PCR assay was used for HMW-MAA and MART-1 mRNA detection.
Expression frequency and immunostaining intensity were higher for HMW-MAA than MART-1in nodal macrometastases (P < 0.0001and P < 0.0001, respectively) and micrometastases (P < 0.0001and P = 0.004, respectively). All 52 (100%) macrometastases were positive with HMW-MAA – specific mAbs, whereas 43 (83%) were positive with MART-1– specific mAbs. In a comparison analysis, 23 of 23 (100%) micrometastases were HMW-MAA – positive, whereas 21 (91%) and18 (78%) specimens were S-100p – and HMB-45– positive, respectively. Quantitative real-time reverse-transcriptase PCR analysis of 48 nodal metastases showed HMW-MAA mRNA detection in SLNs with metastases.
HMW-MAA is more sensitive and specific than MART-1, S-100p, and HMB-45 for IHC-based detection of SLN micrometastases. SLN PEAT – based detection specificity of melanoma micrometastases can be improved by IHC with HMW-MAA – specific mAbs.
PMCID: PMC2686117  PMID: 18519770
3.  Clinical Implications of Micrometastasis Detection in Internal Mammary Nodes of Breast Cancer Patients 
Breast Care  2012;7(3):216-219.
The aim of this study was to assess the efficiency of step-serial sectioning (SSS) combined with hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining in detecting micrometastasis of internal mammary lymph nodes (IMLNs).
Patients and Methods
135 IMLNs from 88 breast cancer patients were re-examined by SSS, combined with either H&E or IHC staining of the biomarkers cytokeratin-19 and epithelial membrane antigen.
Of the 135 IMLNs, 6 nodes from 5 cases displayed 1 or more micrometastases. Histological grade and lymphovascular invasion status were significantly correlated with micrometastasis in the IMLNs (p = 0.018 and 0.001, respectively). Of the 6 nodes positive for micrometastasis, 1 node was detected by both H&E and IHC staining. The remaining 5 nodes from 4 cases showed evident tumor cells only by IHC staining. Finally 8 of the 83 patients (9.64%) without IMLN metastasis showed distant metastasis, while 2 of the 5 patients (40%) with IMLN metastasis showed distant metastasis within 28 months of operation.
SSS combined with H&E and IHC staining is more efficient in detecting micrometastasis than classic routine single-slice H&E only.
PMCID: PMC3409387  PMID: 22872795
Breast cancer; Internal mammary nodes; Micrometastasis; Step-serial sectioning
4.  Lymph node micrometastasis and its correlation with MMP-2 expression in gastric carcinoma 
AIM: To examine matrix metalloproteinase-2 (MMP-2) expression in gastric cancer tissues and to evaluate its relationship with lymph node micrometastasis.
METHODS: The authors studied 850 lymph nodes resected from 30 patients with gastric carcinoma who underwent gastrectomy with lymphadenectomy using reverse transcription polymerase chain reaction (RT-PCR) assay in addition to H-E staining. MMP-2 expression of the tumor tissues was detected by immunohistochemical technique (EliVisionTM plus).
RESULTS: MMP-2 expression was positive in 21 (70%) cases and negative in 9 (30%) cases. No significant correlations were found between MMP-2 expression and other variables such as age, gender, tumor location, tumor diameter, Lauren classification and lymphatic invasion. In contrast, MMP-2 expression correlated significantly with depth of tumor infiltration (P = 0.022), lymph node metastasis (P = 0.030) and tumor differentiation (P = 0.043). Lymph node micrometastases were detected in 77 (12.5%) lymph nodes of 14 (46.7%) gastric carcinoma patients. MMP-2 expression was positive in 12 (85.7%) of the 14 patients with lymph node micrometastasis, and in 9 (56.3%) of the 16 patients without lymph node micrometastasis (P = 0.118).
CONCOUSION: Our results demonstrate that MMP-2 expression has significant correlation with tumor invasion, tumor differentiation and lymph node metastases. MMP-2 expression may be an important biological characteristics and significant prognostic parameter of gastric carcinoma. We also conclude that MMP-2 may participate in the development of lymph node micrometastasis of gastric carcinoma. Further investigations are needed to draw a conclusion.
PMCID: PMC4087816  PMID: 16718824
Gastric carcinoma; Lymph node micrometastasis; MMP-2; RT-PCR; Immunohistochemistry
5.  Calcineurin/NFAT pathway mediates wear particle-induced TNF-α release and osteoclastogenesis from mice bone marrow macrophages in vitro 
Acta Pharmacologica Sinica  2013;34(11):1457-1466.
To investigate the roles of the calcineurin/nuclear factor of activated T cells (NFAT) pathway in regulation of wear particles-induced cytokine release and osteoclastogenesis from mouse bone marrow macrophages in vitro.
Osteoclasts were induced from mouse bone marrow macrophages (BMMs) in the presence of 100 ng/mL receptor activator of NF-κB ligand (RANKL). Acridine orange staining and MTT assay were used to detect the cell viability. Osteoclastogenesis was determined using TRAP staining and RT-PCR. Bone pit resorption assay was used to examine osteoclast phenotype. The expression and cellular localization of NFATc1 were examined using RT-PCR and immunofluorescent staining. The production of TNFα was analyzed with ELISA.
Titanium (Ti) or polymethylmethacrylate (PMMA) particles (0.1 mg/mL) did not significantly change the viability of BMMs, but twice increased the differentiation of BMMs into mature osteoclasts, and markedly increased TNF-α production. The TNF-α level in the PMMA group was significantly higher than in the Ti group (96 h). The expression of NFATc1 was found in BMMs in the presence of the wear particles and RANKL. In bone pit resorption assay, the wear particles significantly increased the resorption area and total number of resorption pits in BMMs-seeded ivory slices. Addition of 11R-VIVIT peptide (a specific inhibitor of calcineurin-mediated NFAT activation, 2.0 μmol/L) did not significantly affect the viability of BMMs, but abolished almost all the wear particle-induced alterations in BMMs. Furthermore, VIVIT reduced TNF-α production much more efficiently in the PMMA group than in the Ti group (96 h).
Calcineurin/NFAT pathway mediates wear particles-induced TNF-α release and osteoclastogenesis from BMMs. Blockade of this signaling pathway with VIVIT may provide a promising therapeutic modality for the treatment of periprosthetic osteolysis.
PMCID: PMC4006473  PMID: 24056707
wear particle; bone marrow macrophage; osteoclast; TNF-α; calcineurin; nuclear factor of activated T cells (NFAT); 11R-VIVIT peptide; arthroplasty; periprosthetic osteolysis; aseptic loosening
6.  Molecular evidence-based use of bone resorption-targeted therapy in prostate cancer patients at high risk for bone involvement. 
Molecular Medicine  2002;8(11):667-675.
BACKGROUND: To improve median survival of patients with prostate cancer that has metastasized to bone, we need to better understand the early events of the metastatic process in skeleton and develop molecular tools capable of detecting the early tumor cell dissemination into bones (micrometastasis stage). However, the initial phase of tumor cell dissemination into the bone marrow is promptly followed by the migration of tumor cells into bone matrix, which is a crucial step that signals the transformation of micrometastasis to macrometastasis stage and clinically evident metastasis. The migration of cancer cells into bone matrix requires the activation of local bone resorption. Such an event contributes to tumor cell hiding/ escaping from high immunologic surveillance of bone marrow cells. Within bone matrix, tumor cells are establishing plethoric cell-cell interactions with bone marrow-residing cells, ensuring their survival and growth. Recently, RT-PCR detections of tumor marker transcripts, such as PSA and PSMA mRNA performed in RNA extracts of peripheral blood nucleated cells and bone marrow biopsy, have enabled the stratification of patients with clinically localized prostate cancer being of high risk for extraprostatic disease and bone involvement. Therefore, it is conceivable that bisphosphonate blockade of bone resorption can inhibit the migration of tumor cells into bone matrix during the early phase of disease dissemination into bone marrow (micrometastasis stage). Consequently, assessment of the efficacy and efficiency of bisphosphonates to arrest the evolution of bone lesions in this particular clinical setting of patients with clinically localized prostate cancer and positive molecular staging status (high risk for bone involvement) is warranted.
PMCID: PMC2039951  PMID: 12520083
7.  Effect of Androgen Blockade on HER-2 and Matrix Metalloproteinase-2 Expression on Bone Marrow Micrometastasis and Stromal Cells in Men with Prostate Cancer 
The Scientific World Journal  2013;2013:281291.
Introduction. HER-2 has been associated with castrate resistant prostate cancer and matrix metalloproteinase-2 (MMP-2) in the dissemination and invasion of tumor cells as well as activating angiogenesis. We present an immunocytochemical study of the effect of androgen blockade on the expression of HER-2 and MMP-2 in bone marrow micrometastasis and the surrounding stromal cells in men with prostate cancer. Methods and Patients. A cross-sectional study of men with prostate cancer. Touch preps were obtained from bone marrow biopsies of men with prostate cancer, before and after radical prostatectomy and during androgen blockade. Micrometastasis detected with anti-PSA immunocytochemistry underwent processing with anti-HER-2 and anti-MMP-2 immunocytochemistry. Patients were defined as HER-2 positive or negative, MMP-2 negative or an MMP-2 pattern described as border or central and stromal MMP-2 defined as positive or negative. The expression of the biomarkers was compared before and after primary treatment and during androgen blockade in relation to the serum PSA at the time of sampling and duration of androgen blockade. Results. 191 men participated, 35 men before surgery and 43 after surgery; there were no significant differences in HER-2 expression between groups, there was no MMP-2 expression centrally or stromal expression of MMP-2. In men with androgen blockade, HER-2 expression was significantly higher; there was a trend for increasing HER-2 expression up to 5 years; central MMP-2 expression significantly increased after 3 years, while stromal MMP-2 significantly increased after 6 years. MMP-2 expression both in micrometastasis and stroma was significantly associated with HER-2 expression. Expression of MMP-2 at the border of the micrometastasis was not associated with HER-2 expression and occurred in the absence of androgen blockade. Conclusions. Androgen blockade decreases serum PSA by eliminating HER-2 negative prostate cancer cells. However, there is early selection of HER-2 positive cancer cells which leads to androgen independence and to increased expression of MMP-2 activity in the micrometastasis. The increased MMP-2 activity in the micrometastasis increases the expression of MMP-2 in the surrounding stromal cells and thus could promote angiogenesis and tumor growth resulting in macrometastatic androgen independent disease.
PMCID: PMC3666220  PMID: 23766685
8.  The significance of clinicopathological aspects of tumor for the detection of liver micrometastasis in patients with colorectal cancer 
Colorectal tumor is one of the main causes of death in our country. The aim of the present study was to determine the clinicopathological aspects of tumor and the presence of hepatic micrometastasis in patients with colorectal cancer (CRC).
Materials and Methods:
Forty two patients with CRC were evaluated in the study surgical treatment was performed and liver biopsy was taken for the evaluation of micrometastasis by immunohistochemistry and polymerase chain reaction. The variables that have been evaluated were: Patient's gender, patients age at the time of diagnosis, size and location of tumor, tumor-node-metastasis stage and grade of the primary tumor, lymph node involvement, lymphovascular and neural invasion, presence of macrometastasis and carcinoembryonic antigen level prior to surgery. After 1 year patients were called and asked to come back to the clinic for elective colonoscopy to evaluate the surgical site for recurrence of tumor and survival. All variables were compared between patients in whom liver micrometastasis were present in comparison with patients without liver micrometastasis.
Of the studied patients (6 with positive micrometatsis and 36 without micrometstasis), 38 were alive after 1 year (6 with positive micrometatsis and 32 without micrometstasis) and the difference was not significant between groups with or without micrometastasis (P = 0.52). In four of survived patients colonoscopy was abnormal, however this difference was not also significant between groups (P = 0.59).
Clinicopathologic aspect of tumor was not different in CRC patients with and without hepatic micrometastasis.
PMCID: PMC4116571  PMID: 25097622
Colorectal cancer; immunohistochemistry; micrometastasis; polymerase chain reaction
9.  Clinical Implications of Immunohistochemically Demonstrated Lymph Node Micrometastasis in Resectable Pancreatic Cancer 
Journal of Korean Medical Science  2011;26(7):881-885.
The purpose of this study was to determine the clinical significance of nodal micrometastasis detected by immunohistochemistry in patients that had undergone curative surgery for pancreatic cancer. Between 2005 and 2006, a total of 208 lymph nodes from 48 consecutive patients with pancreatic cancer that had undergone curative resection were immunostained with monoclonal antibody against pan-ck and CK-19. Micrometastasis was defined as metastasis missed by a routine H&E examination but detected during an immunohistochemical evaluation. Relations between immunohistochemical results and clinical and pathologic features and patient survival were examined. Nodal micrometastases were detected in 5 (29.4%) patients of 17 pN0 patients. Nodal micrometastasis was found to be related to tumor relapse (P = 0.043). Twelve patients without overt nodal metastasis and micrometastasis had better prognosis than 5 patients with only nodal micrometastasis (median survival; 35.9 vs 8.6 months, P < 0.001). The Cox proportional hazard model identified nodal micrometastasis as significant prognostic factors. Although the number of patients with micrometastasis was so small and further study would be needed, our study suggests that the lymph node micrometastasis could be the predictor of worse survival and might indicate aggressive tumor biology among patients undergoing curative resection for pancreas cancer.
PMCID: PMC3124717  PMID: 21738340
Pancreas; Adenocarcinoma; Lymph Nodes; Micrometastasis; Prognosis
10.  Differential regulation of cytokine and cytokine receptor mRNA expression upon infection of bone marrow-derived macrophages with Listeria monocytogenes. 
Infection and Immunity  1996;64(9):3475-3483.
Cytokine and cytokine receptor mRNA expression was analyzed by PCR-assisted amplification of RNA extracted from bone marrow-derived macrophages (BMM phi) at different time points after infection with Listeria monocytogenes. The mRNAs for the cytokines interleukin-1 alpha (IL-1 alpha), IL-1 beta, and tumor necrosis factor alpha (TNF-alpha) were induced early after infection, whereas IL-6 mRNA appeared later and even nonhemolytic Listeria strains, which are unable to grow inside eukaryotic cells, induced the same cytokine mRNAs at levels similar to those of the wild-type strain. In most cases, the amounts of cytokines determined by various bioassays correlated with the level of mRNA induction. Inhibition of phagocytic uptake of L. monocytogenes by cytochalasin D treatment resulted in adherent bacteria which still induced the proinflammatory cytokines. In BMM phi, the level of IL-1 receptor II mRNA was unaffected, whereas mRNA expression of the two subtypes of tumor necrosis factor receptors (TNF-RI and TNF-RII) was differentially regulated upon infection: transcription of TNF-RI was reduced, and that of TNF-RII mRNA was induced. Similar to the decreased TNF-RI mRNA expression, gamma interferon receptor mRNA was downregulated in L. monocytogenes-infected BMM phi. This dose- and time-dependent induction or downregulation of cytokine receptor mRNA following L. monocytogenes infection of BMM phi was not observed upon infection of established macrophage-like cell lines J774 and P388D1. Induction of IL-6 mRNA as well as IL-1 alpha/beta and TNF-alpha mRNAs upon L. monocytogenes infection of BMM phi occurs independently of autocrine TNF-alpha signaling via TNF-RI or TNF-RII, as shown by infection of TNF-RI- and TNF-RII-deficient macrophages derived from mutant B6 x 129 mice. In contrast to gamma interferon receptor mRNA, both TNF receptor subtype mRNAs were not influenced by L. monocytogenes infection of hybrid (B6 x 129) mouse macrophages. Whereas the proinflammatory cytokine mRNAs were even induced after infection with the nonpathogenic species L. innocua, no alteration of cytokine receptor mRNA expression was observed after challenge of BMM phi with this nonpathogenic species, suggesting that the modulation of cytokine and cytokine receptor expression by L. monocytogenes could be an important way of inhibition of macrophage stimulation.
PMCID: PMC174251  PMID: 8751887
11.  Detection of micrometastasis in peripheral blood by multi-sampling in patients with colorectal cancer 
AIM: To evaluate the reverse transcriptase-PCR assay and multiple sampling for detection of cytokeratin-positive cells in peripheral blood of colorectal carcinoma patients and to investigate the clinical significance of micrometastasis in peripheral blood.
METHODS: The expression of CK20 mRNA by RT-PCR was investigated in bone marrow, portal vein and peripheral blood in 58 colorectal cancer patients and 12 controls without known cancer. The peripheral blood was sampled twice at intervals of 3 d before operation. All the patients were followed up for one year.
RESULTS: There was no positive expression of CK20mRNA in 12 volunteers. The positive expression of CK20mRNA was 77.6% (45/58) in bone marrow, and that in portal vein was 74.1% (43/58) of colorectal carcinoma patients. The positive expression of CK20mRNA cells in peripheral blood rose from 44.8% (26/58) to 69.0% (40/58) (P<0.01). The total positivity of CK20mRNA expression in peripheral blood was similar to the positivity of CK20mRNA in bone marrow and portal vein. The positive rates became higher in later clinical stages than in early stages. The CK20mRNA positive patients had a higher relapse rate within one year than the CK20mRNA negative patients.
CONCLUSION: Multiple blood sampling can increase the detection of tumor cells in peripheral blood by RT-PCR for CK20mRNA in colorectal carcinoma patients and it is as sensitive and specific as that of bone marrow and portal vein. This technique may be reliable and convenient to diagnose micrometastasis of colorectal carcinoma and has an important significance in determining the prognosis of cancer patients.
PMCID: PMC4205357  PMID: 15637763
Colorectal Cancer; CK20mRNA; Micrometastasis
12.  Calcitonin gene-related peptide stimulates stromal cell osteogenic differentiation and inhibits RANKL induced NF-κB activation, osteoclastogenesis and bone resorption 
Bone  2009;46(5):1369-1379.
Previously we observed that capsaicin treatment in rats inhibited sensory neuropeptide signaling, with a concurrent reduction in trabecular bone formation and bone volume, and an increase in osteoclast numbers and bone resorption. Calcitonin gene-related peptide (CGRP) is a neuropeptide richly distributed in sensory neurons innervating the skeleton and we postulated that CGRP signaling regulates bone integrity. In this study we examined CGRP effects on stromal and bone cell differentiation and activity in vitro. CGRP receptors were detected by immunocytochemical staining and real time PCR assays in mouse bone marrow stromal cells (BMSCs) and bone marrow macrophages (BMMs). CGRP effects on BMSC proliferation and osteoblastic differentiation were studied using BrdU incorporation, PCR products, alkaline phosphatase (ALP) activity, and mineralization assays. CGRP effects on BMM osteoclastic differentiation and activity were determined by quantifying tartrate-resistant acid phosphatase positive (TRAP+) multinucleated cells, pit erosion area, mRNA levels of TRAP and cathepsin K, and nuclear factor-κB (NF-κB) nuclear localization. BMSCs, osteoblasts, BMMs, and osteoclasts all expressed CGRP receptors. CGRP (10-10-10-8M) stimulated BMSC proliferation, up-regulated the expression of osteoblastic genes, and increased ALP activity and mineralization in the BMSCs. In BMM cultures CGRP (10-8M) inhibited receptor activator of NF-κB ligand (RANKL) activation of NF-κB. CGRP also down-regulated osteoclastic genes like TRAP and cathepsin K, decreased the numbers of TRAP+ cells, and inhibited bone resorption activity in RANKL stimulated BMMs. These results suggest that CGRP signaling maintains bone mass both by directly stimulating stromal cell osteoblastic differentiation and by inhibiting RANKL induced NF-κB activation, osteoclastogenesis, and bone resorption.
PMCID: PMC2854244  PMID: 19962460
Neuropeptide; Calcitonin gene-related protein; Bone marrow stromal cell; Osteoblast; Osteoclasts; Mineralization
13.  Prognostic Relevance of Occult Nodal Micrometastases and Circulating Tumor Cells in Colorectal Cancer in a Prospective Multicenter Trial 
Nodal micrometastasis and circulating tumor cells (CTC) detected by multimarker quantitative real-time RT-PCR (qRT) may have prognostic importance in patients with colorectal cancer (CRC).
Experimental Design
Paraffin-embedded sentinel lymph nodes (SLNs) from 67 patients and blood from 34 of these patients was evaluated in a prospective multicenter trial of SLN mapping in CRC. SLNs were examined by hematoxylin and eosin staining (H&E) and cytokeratin immunohistochemistry (CK-IHC). Both SLNs and blood were examined by a four-marker qRT assay (c-MET, MAGE-A3, GalNAc-T, CK20); qRT results were correlated with disease stage and outcome.
In H&E SLN negative patients that recurred, CK-IHC and qRT detected metastasis in 30% and 60% of patients, respectively. DFS differed significantly by multimarker qRT upstaged SLN (p=0.014). qRT analysis of blood for CTC correlated with overall survival (p=0.040).
Molecular assessment for micrometastasis in SLN and blood specimens may help identify patients at high-risk for recurrent CRC, who could benefit from adjuvant therapy.
PMCID: PMC2586882  PMID: 19010855
RT-PCR; colorectal cancer; micrometastasis; sentinel lymph node
14.  Echogenic perfluorohexane-loaded macrophages adhere in vivo to activated vascular endothelium in mice, an explorative study 
Macrophages may concentrate ultrasound contrast agents and exhibit selective adhesion to activated endothelium. The present study investigates in mice the potential of perfluorohexane (PFH) loaded macrophages to act as ultrasound contrast agent with high reflectivity and specifically targeted at (atherosclerotic) vascular lesions.
Lung passage was evaluated with a mouse echo scanner after injection, at a slow pace or as a bolus, of varying doses of PFH-loaded and unloaded bone marrow macrophages (BMM) into the jugular vein. The interaction of PFH-loaded and unloaded BMM with TNF-α stimulated carotid artery endothelium after tail vein injection was assessed by means of intravital microscopy.
High doses of jugular vein injected PFH-loaded BMM were visible with ultrasound in the pulmonary artery and detectable in the carotid artery. At intravital microscopy, tail vein injected BMM exhibited rolling and adhesion behavior at the TNF-α stimulated carotid endothelium, similar to that of native blood leukocytes. Rolling behavior was not different between PFH-loaded and unloaded BMM (p = 0.38).
In vivo, perfluorohexane loaded macrophages pass the pulmonary circulation and appear on the arterial side. Moreover, they roll and adhere selectively to activated endothelium under physiological flow conditions. These findings indicate that perfluorohexane loaded BMM could be used to study processes in vivo where endothelial activation plays a role, such as atherosclerosis.
PMCID: PMC4293794  PMID: 25567641
Atherosclerosis; Ultrasound contrast; Macrophage; Endothelium; Perfluorocarbon
15.  Comparative Assessment of Lymph Node Micrometastasis in Cervical, Endometrial and Vulvar Cancer: Insights on the Real Time qRT-PCR Approach versus Immunohistochemistry, Employing Dual Molecular Markers 
BioMed Research International  2014;2014:187684.
To address the value of qRT-PCR and IHC in accurately detecting lymph node micrometastasis in gynecological cancer, we performed a systematic approach, using a set of dual molecular tumor-specific markers such as cytokeratin 19 (CK19) and carbonic anhydrase 9 (CA9), in a series of 46 patients (19 with cervical cancer, 18 with endometrial cancer, and 9 with vulvar cancer). A total of 1281 lymph nodes were analyzed and 28 were found positive by histopathology. Following this documentation, 82 lymph nodes, 11 positive and 71 negative, were randomly selected and further analyzed both by IHC and qRT-PCR for CK19 and CA9 expression. All 11 (100%) expressed CK19 by IHC, while only 6 (54.5%) expressed CA9. On the contrary, all the histologically negative for micrometastases lymph nodes were also negative by IHC analysis for both markers. The comparative diagnostic efficacy of the two markers using qRT-PCR, however, disclosed that the analysis of the same aliquots of the 82 lymph nodes led to 100% specificity for the CK19 biomarker, while, in contrast, CA9 failed to recapitulate a similar pattern. These data suggest that qRT-PCR exhibits a better diagnostic accuracy compared to IHC, while CK19 displays a consistent pattern of detection compared to CA9.
PMCID: PMC3910066  PMID: 24527437
16.  Expression of human mammaglobin as a marker of bone marrow micrometastasis in breast cancer 
The aim of the present study was to detect the expression of human mammaglobin (hMAM) mRNA in the bone marrow (BM) of patients with breast cancer and determine the relationship between micrometastasis and clinicopathological parameters as well as selected molecular markers and breast cancer prognosis. The expression of hMAM mRNA in the BM of patients with breast cancer was determined by RT-PCR. The expression of ER, PR and Cath-D in cancer tissues was detected by immunohistochemistry. A positive expression rate for hMAM of 38.2% in 102 patients with stage I–III breast cancer was found. The expression of hMAM was higher in patients with T2–3 (>2 cm) tumors than in those with T1 tumors (≤2 cm) (χ2=19.20, P=0.001) and in patients with stage II or III tumors than in patients with stage I tumors (χ2=15.101, P=0.001). The expression of hMAM in the BM of breast cancer patients categorized as grade 1 was lower than that in those of grade 2 or 3 (χ2=8.522, P=0.014), and hMAM expression was related to the pathological type of tumor (χ2=6.892, P=0.032) and the degree of axillary lymph node metastasis (χ2=14.050, P=0.001). The expression of hMAM in BM was much higher in patients with ER(−) or ER(+) tumors than in those with ER(++ or +++) (χ2=11.800, P=0.003), and those with PR (χ2=8.759, P=0.013). hMAM expression in BM was also significantly positively correlated with Cath-D expression (χ2=6.623, P=0.036). However, no correlation was found between hMAM expression and patient age (χ2=1.056, P=0.304). There was a strong correlation between patients with positive expression of hMAM in the BM and the presence of distant metastases (P=0.009). In conclusion, micrometastasis in the BM correlates with certain clinical pathological parameters and several tumor markers. Patients with positive expression of hMAM in the BM have a greater chance of distant metastasis and poor prognosis. The detection of micrometastasis may be one of the most advantageous markers for predicting the prognosis of breast cancer.
PMCID: PMC3438616  PMID: 22969928
breast cancer; bone marrow micrometastasis; human mammaglobin; biological factor; RT-PCR; immunohistochemistry; prognosis
17.  Comparative evaluation of cell-free tumor DNA in blood and disseminated tumor cells in bone marrow of patients with primary breast cancer 
The origin and clinical relevance of circulating cell-free tumor DNA in the blood of cancer patients is still unclear. Here we investigated whether the detection of this DNA is related to bone marrow (BM) micrometastasis and tumor recurrence in breast cancer patients.
BM aspirates of 81 primary breast cancer patients were analyzed for the presence of disseminated tumor cells (DTC) by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3. PCR-based fluorescence microsatellite analysis was performed for detection of loss of heterozygosity (LOH) at 6 polymorphic markers using cell-free serum DNA. The data were correlated with established risk factors, and patients were followed-up over 6-10 years.
LOH was detected in 33.5% of blood samples. The occurrence of LOH at the entire microsatellite marker set correlated with histopathology (P = 0.05) and grading (P = 0.006) of the primary tumor. The genomic region characterized by marker D9S171 was only affected by LOH in patients with increased tumor stages (pT2-4, P < 0.05) and older age (≥ 55 years, P = 0.05). Kaplan-Meier analysis showed that LOH at D3S1255 (P = 0.009) and D9S171 (P = 0.001) were significantly associated with tumor relapse. In BM, DTC were detected in 39.5% of the patients, and this finding correlated with distant metastases (P < 0.05). Patients with DTC-positive BM had higher DNA yields in their blood than patients with DTC-negative BM (P < 0.05). However, no significant correlations were found between the presence of DTC in BM and the detection of marker-specific LOH on blood DNA.
The detection of LOH on cell-free tumor DNA in blood is unrelated to BM micrometastasis and provides independent information on breast cancer progression.
PMCID: PMC2790848  PMID: 19772563
18.  Rate of para-aortic lymph node micrometastasis in patients with locally advanced cervical cancer 
Gynecologic oncology  2010;119(3):422-425.
Patients with micrometastasis to para-aortic lymph nodes may benefit from extended field chemoradiation. To determine the rate of para-aortic node micrometastasis in patients with locally advanced cervical cancer undergoing laparoscopic extraperitoneal para-aortic lymphadenectomy
We prospectively identified consecutive patients diagnosed with stage IB2-IVA biopsy-proven cervical cancer. Eligible patients included those who were candidates for treatment with radiotherapy and concurrent chemotherapy and had no evidence of para-aortic lymphadenopathy (all lymph nodes < 2 cm in diameter) by preoperative computed tomography or magnetic resonance imaging. All patients underwent preoperative positron emission tomography/computed tomography and laparoscopic extraperitoneal para-aortic lymphadenectomy. All lymph nodes were assessed for metastasis by routine hematoxylin-eosin (H&E) staining. Ultrastaging (serial sectioning) and immunohistochemical analysis were performed in H&E-negative specimens.
Thirteen (22%) of 60 consecutive patients had para-aortic lymph node metastases detected on routine H&E staining. Of the remaining 47 patients, one (2.1%) had evidence of micrometastasis, which was detected by ultrastaging. This patient completed whole pelvic radiotherapy and chemotherapy but had a recurrence 27 months after completion of therapy.
The rate of para-aortic node micrometastasis in patients with locally advanced cervical cancer is low. The role of routine ultrastaging and immunohistochemical analysis in such patients remains uncertain. Future studies are needed to determine the clinical impact of para-aortic node micrometastasis in patients with locally advanced cervical cancer.
PMCID: PMC4286254  PMID: 20837355
19.  Substance P stimulates bone marrow stromal cell osteogenic activity, osteoclast differentiation, and resorption activity in vitro 
Bone  2009;45(2):309-320.
SP is a neuropeptide distributed in the sensory nerve fibers that innervate the medullar tissues of bone, as well as the periosteum. Previously we demonstrated that inhibition of neuropeptide signaling after capsaicin treatment resulted in a loss of bone mass and we hypothesized that SP contributes to bone integrity by stimulating osteogenesis.
Materials and Methods
Osteoblast precursors (bone marrow stromal cells, BMSCs) and osteoclast precursors (bone marrow macrophages, BMMs) derived from C57BL/6 mice were cultured. Expression of the SP receptor (NK1) was detected by using immunocytochemical staining and PCR. Effects of SP on proliferation and differentiation of BMSCs were studied by measuring BrdU incorporation, gene expression, alkaline phosphatase activity, and osteocalcin and Runx2 protein levels with EIA and western blot assays, respectively. Effects of SP on BMMs were determined using a BrdU assay, counting multinucleated cells staining positive for tartrate-resistant acid phosphatase (TRAP+), measuring pit erosion area, and evaluating RANKL protein production and NF-κB activity with ELISA and western blot.
The NK1 receptor was expressed in both BMSCs and BMMs. SP stimulated the proliferation of BMSCs in a concentration-dependent manner. Low concentrations (10−12 M) of SP stimulated alkaline phosphatase and osteocalcin expression, increased alkaline phosphatase activity, and up-regulated Runx2 protein levels, and higher concentrations of SP (10−8 M) enhanced mineralization in differentiated BMSCs. SP also stimulated BMSCs to produce RANKL, but at concentrations too low to evoke osteoclastogenesis in co-culture with macrophages in the presence of SP. SP also activated NF-κB in BMMs and directly facilitate RANKL induced macrophage osteoclastogenesis and bone resorption activity.
NK1 receptors are expressed by osteoblast and osteoclast precursors and SP stimulates osteoblast and osteoclast differentiation and function in vitro. SP neurotransmitter release from sensory neurons could potentially regulate local bone turnover in vivo.
PMCID: PMC2706279  PMID: 19379851
Neuropeptide; Substance P; Bone Marrow Stromal Cell; Mouse; Osteogenesis; Osteoblast; Osteoclasts
20.  Cell Adhesion Signaling Regulates RANK Expression in Osteoclast Precursors 
PLoS ONE  2012;7(11):e48795.
Cells with monocyte/macrophage lineage expressing receptor activator of NF-κB (RANK) differentiate into osteoclasts following stimulation with the RANK ligand (RANKL). Cell adhesion signaling is also required for osteoclast differentiation from precursors. However, details of the mechanism by which cell adhesion signals induce osteoclast differentiation have not been fully elucidated. To investigate the participation of cell adhesion signaling in osteoclast differentiation, mouse bone marrow-derived macrophages (BMMs) were used as osteoclast precursors, and cultured on either plastic cell culture dishes (adherent condition) or the top surface of semisolid methylcellulose gel loaded in culture tubes (non-adherent condition). BMMs cultured under the adherent condition differentiated into osteoclasts in response to RANKL stimulation. However, under the non-adherent condition, the efficiency of osteoclast differentiation was markedly reduced even in the presence of RANKL. These BMMs retained macrophage characteristics including phagocytic function and gene expression profile. Lipopolysaccharide (LPS) and tumor necrosis factor –αTNF-α activated the NF-κB-mediated signaling pathways under both the adherent and non-adherent conditions, while RANKL activated the pathways only under the adherent condition. BMMs highly expressed RANK mRNA and protein under the adherent condition as compared to the non-adherent condition. Also, BMMs transferred from the adherent to non-adherent condition showed downregulated RANK expression within 24 hours. In contrast, transferring those from the non-adherent to adherent condition significantly increased the level of RANK expression. Moreover, interruption of cell adhesion signaling by echistatin, an RGD-containing disintegrin, decreased RANK expression in BMMs, while forced expression of either RANK or TNFR-associated factor 6 (TRAF6) in BMMs induced their differentiation into osteoclasts even under the non-adherent condition. These results suggest that cell adhesion signaling regulates RANK expression in osteoclast precursors.
PMCID: PMC3490906  PMID: 23139818
21.  The tumor necrosis factor alpha-stimulating region of galactose-inhibitable lectin of Entamoeba histolytica activates gamma interferon-primed macrophages for amebicidal activity mediated by nitric oxide. 
Infection and Immunity  1997;65(7):2522-2527.
Entamoeba histolytica adheres via galactose-lectin (Gal-lectin) to human colonic mucins and intestinal epithelial cells as a prerequisite to amebic invasion. Native Gal-lectin is a protective antigen in the gerbil model of amebiasis. Amino acids 596 to 1082 of Gal-lectin mediate E. histolytica adherence to target cells and stimulate tumor necrosis factor alpha (TNF-alpha) production by naive murine bone marrow macrophages (BMM). Resistance to amebiasis requires an effective cell-mediated immune response against E. histolytica trophozoites mediated by nitric oxide (NO) released from activated macrophages. Herein, we determine whether the TNF-alpha-stimulating region of Gal-lectin can activate gamma interferon (IFN-gamma)-primed BMM for NO production and amebicidal activity. Native Gal-lectin (100 to 500 ng/ml) stimulated TNF-alpha and inducible nitric oxide synthase (iNOS) mRNA expression in IFN-gamma-primed BMM as did lipopolysaccharide (100 ng/ml). Primed BMM produced TNF-alpha and NO in response to Gal-lectin in a dose-dependent manner. Antilectin monoclonal antibody IG7, which recognizes a domain (amino acids 596 to 818) of the TNF-alpha mRNA-stimulating region of Gal-lectin, specifically inhibited TNF-alpha and iNOS mRNA induction and TNF-alpha and NO production by primed BMM in response to Gal-lectin (100 ng/ml). Simultaneous treatment of BMM with IFN-gamma and Gal-lectin (100 ng/ml) activated the cells to kill E. histolytica trophozoites, whereas IFN-gamma treatment alone had no effect. In the presence of monoclonal antibody 1G7 or aminoguanidine (an iNOS inhibitor), NO production and amebicidal activity were inhibited >80%. These results suggest that the TNF-alpha-stimulating region of native Gal-lectin is a potent stimulus of IFN-gamma-primed BMM for NO production, which is essential for host defense against amebiasis.
PMCID: PMC175356  PMID: 9199414
22.  Reactivity of rat bone marrow-derived macrophages to neurotransmitter stimulation in the context of collagen II-induced arthritis 
Numerous observations indicate that rheumatoid arthritis (RA) has a bone marrow component. In parallel, local synovial changes depend on neuronal components of the peripheral sympathetic nervous system. Here, we wanted to analyze whether collagen II-induced arthritis (CIA) has an impact on number, adhesion, apoptosis, and proliferation of the macrophage subset of bone marrow cells and how alterations in neurotransmitter microenvironment affect these properties.
Bone marrow-derived macrophages (BMMs) were isolated from Dark Agouti rats at different stages of CIA, and number, adhesion, caspase 3/7 activity, and proliferation were analyzed in the presence of acetylcholine (ACh), noradrenaline (NA), and vasoactive intestinal peptide (VIP).
Opposed to enhanced CD11b+ (cluster of differentiation 11b-positive) and EMR1+ (epidermal growth factor-like module-containing mucin-like hormone receptor-like 1-positive) cells, characterizing the macrophage subset, in native bone marrow of rats with acute inflammatory arthritis, we found decreased numbers of CIA macrophages after enrichment and culture in comparison with healthy (control) animals. Adhesion studies revealed significantly reduced attachment to plastic in acute arthritis and collagen type I and fibronectin in chronic arthritis. Additionally, we found a strong reduction in proliferation of BMMs at CIA onset and in the chronic phase of CIA. Apoptosis remained unaffected. Neurotransmitter stimulation profoundly affected proliferation, adhesion, and apoptosis of BMMs from CIA and control rats, depending on disease time point. Cultured BMMs from CIA and control animals expressed neurotransmitter receptors for ACh, VIP and NA, but the expression profile seemed not to be affected by CIA.
Induction of CIA distinctly inhibits proliferation of BMMs in low- and non-inflammatory phases and reduces attachment to plastic at the acute inflammatory arthritis stage and adhesion to collagen I and fibronectin at the chronic stage. Influence of neurotransmitter stimulation on adhesion, apoptosis, and proliferation is altered by CIA depending on disease stage. We suggest an altered reactivity of BMMs to neurotransmitter stimulation caused by CIA and maybe also by aging.
Electronic supplementary material
The online version of this article (doi:10.1186/s13075-015-0684-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4496866  PMID: 26104678
23.  Comparison of Immunohistochemistry with Conventional Histopathology for Evaluation of Sentinel Lymph Node in Breast Cancer 
The Indian Journal of Surgery  2010;73(2):107-110.
The best method of pathological evaluation of sentinel lymph node in breast cancer has not been agreed upon. Immunohistochemical (IHC) techniques have shown a greater sensitivity over conventional histology for the detection of micrometastais. The aim of the study was to determine whether IHC for Epithelial Membrane Antigen (EMA) on the sentinel node could be more sensitive than conventional histology for diagnosing micrometastasis in sentinel lymph nodes. Eighty-four clinically node negative breast cancer patients underwent sentinel node biopsy at time of surgery for breast cancer. The node was subjected to conventional histopathology as well as IHC for EMA. The sensitivity of histology viz a viz IHC for EMA for detection of sentinel node metastasis was 88% and the specitficity was 96%. The overall diagnostic accuray of histology viz a viz IHC was 93%. There were 4 patients with micrometastasis (<2.0 mm), which were positive on IHC but negative on histology. Two patients with poorly differentiated breast cancer had a false negative IHC for EMA result as compared to histology. Immunohistochemistry for Epithelial Membrane Antigen can increase the detection rate of micrometastasis in sentinel lymph node. This can have important bearing on deciding the need of adjuvant systemic therapy. A false negative result for EMA may be seen in patients with poorly differential cancer. Therefore the best policy seems to employ both histopathology and IHC for EMA for the comprehensive evaluation of sentinel lymph node in breast cancer.
PMCID: PMC3077160  PMID: 22468058
Sentinel node; Breast cancer; Immunohistochemistry
24.  Multiparameter flow cytometry as a tool for the detection of micrometastatic tumour cells in the sentinel lymph node procedure of patients with breast cancer 
Journal of Clinical Pathology  2002;55(5):359-366.
Aim: To investigate whether multiparameter flow cytometry (MP-FCM) can be used for the detection of micrometastasis in sentinel lymph nodes (SLNs) in breast cancer.
Methods: Formalin fixed, paraffin wax embedded sentinel lymph nodes (n = 238) from 98 patients were analysed. For each lymph node, sections for haematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) for cytokeratin (MNF116) were cut at three levels with a distance of 500 μm. The intervening material was used for MP-FCM. Cells were immunostained with MNF116, followed by an incubation with fluorescein isothiocyanate (FITC) labelled goat antimouse immunoglobulin. DNA was stained using propidium iodide. From each lymph node 100 000 cells were analysed on the flow cytometer.
Results: Thirty eight of the 98 patients with breast carcinoma showed evidence of metastatic disease in the SLN by one ore more of the three methods. In 37 of 38 cases where metastatic cells were seen in the routine H&E and/or IHC, more than 1% cytokeratin positive cells were detected by MP-FCM. In 24 patients, metastatic foci were more than 2 mm (macrometastasis) and in 14 these foci were smaller than 2 mm (micrometastasis). In three of these 14 cases, MP-FCM revealed positive SLNs, although this was not seen at first glance in the H&E or IHC sections. After revision of the slides, one of these three remained negative. However, MP-FCM analysis of the cytokeratin positive cells showed an aneuploid DNA peak, which was almost identical to that of the primary breast tumour. Duplicate measurements, done in 41 cases, showed a 99% reproducibility. In five of 14 patients with micrometastasis, one or two metastatic foci were found in the non-SLN. However, in 15 of 24 macrometastases multiple non-SLNs were found to have metastatic tumour. All micrometastases except for the remaining negative one mentioned above showed only diploid tumour cells, despite the fact that their primary tumours contained both diploid and aneuploid tumour cells. In primary tumours with more than 60% aneuploid cells, predominantly aneuploid macrometastasis were found, whereas diploid primary tumours only showed diploid micrometastases or macrometastases in their SLN. Aneuploid SLN macrometastases were associated with non-SLN metastases in five of seven patients, whereas diploid cases showed additional non-SLN metastases in only seven of 16 patients.
Conclusion: In all cases, MP-FCM was sufficient to detect micrometastatic tumour cells in a large volume of lymph node tissue from SLNs. In some cases it was superior to H&E and IHC staining. Approximately 30% of SLN micrometastases are accompanied by additional non-SLN metastases. The size of the aneuploid fraction (> 60%) in the primary tumour may influence the risk of having both SLN and non-SLN metastases.
PMCID: PMC1769646  PMID: 11986342
breast cancer; sentinel node; flow cytometry; double staining
25.  Sentinel Lymph Node Biopsy in the Management of Early-Stage Cervical Carcinoma 
Gynecologic oncology  2011;120(3):347-352.
We aimed to determine the sentinel lymph node detection rates, accuracy in predicting the status of lymph node metastasis, and if pathologic ultrastaging improves the detection of micrometastases and isolated tumor cells at the time of primary surgery for cervical cancer.
A prospective, non-randomized study of women with early-stage (FIGO stage IA1 with lymphovascular space involvement – IIA) cervical carcinoma was conducted from June 2003 to August 2009. All patients underwent an intraoperative intracervical blue dye injection. Patients who underwent a preoperative lymphoscintigraphy received a 99m Tc sulfur colloid injection in addition. All patients underwent sentinel lymph node (SLN) identification followed by a complete pelvic node and parametrial dissection. SLN were evaluated using our institutional protocol that included pathologic ultrastaging.
SLN mapping was successful in 77 (95%) of 81 patients. A total of 316 SLN were identified, with a median of 3 SLN per patient (range, 0-10 SLN). The majority (85%) of SLN were located at three main sites: the external iliac (35%); internal iliac (30%); and obturator (20%). Positive lymph nodes (LN) were identified in 26 (32%) patients, including 21 patients with positive SLN. Fifteen of 21 patients (71%) had SLN metastasis detected on routine processing. SLN ultrastaging detected metastasis in an additional 6/21 patients (29%). Two patients had grossly positive LN at exploration, and mapping was abandoned. Three of 26 (12%) patients had successful SLN mapping; however, the SLN failed to identify the metastatic LN. Of these 3 false negative cases, 2 patients had a metastatic parametrial node as the only positive LN with multiple negative pelvic nodes including negative SLN. One patient with stage IA1 disease and lymphovascular invasion had unilateral SLN mapping and a metastatic common iliac LN identified on completion lymphadenectomy of the contralateral side that did not map. The 4 (5%) patients with unsuccessful mapping included 1 who had grossly positive nodes identified at the time of laparotomy; the remaining 3 occurred during each surgeon's initial SLN mapping learning phase.
SLN mapping in early-stage cervical carcinoma yields high detection rates. Ultrastaging improves micrometastasis detection. Parametrectomy and side-specific lymphadenectomy (in cases of failed mapping) remain important components of the surgical management of select cases.
PMCID: PMC3951119  PMID: 21216450
Sentinel lymph nodes; micrometastasis; cervical cancer

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