Diagnosis Of Cancer Metastasis Research Articles

Tracking Cancer Metastasis In Vivo by Using an Iridium-Based Hypoxia-Activated Optical Oxygen Nanosensor.

We have developed a nanosensor for tracking cancer metastasis by noninvasive real-time whole-body optical imaging. The nanosensor is prepared by the formation of co-micelles from a poly(N-vinylpyrrolidone)-conjugated iridium(III) complex (Ir-PVP) and poly(ε-caprolactone)-b-poly(N-vinylpyrrolidone) (PCL-PVP). The near-infrared phosphorescence emission of the nanosensor could be selectively activated in the hypoxic microenvironment induced by cancer cells. The detection ability of the nanosensor was examined in cells and different animal models. After intravenous injection, the nanosensor can be effectively delivered to the lung and lymph node, and cancer cell metastasis through bloodstream or lymphatics can be quickly detected with high signal-to-background ratio by whole-body imaging and organ imaging. Moreover, the nanosensor exhibits good biocompatibility both in vitro and in vivo. The nanosensor is believed to be a powerful tool for the diagnosis of cancer metastasis.

LASS2/TMSG1 inhibits growth and invasion of breast cancer cell in vitro through regulation of vacuolar ATPase activity.

Homo sapiens longevity assurance homologue 2 of yeast LAG1 (LASS2)/tumor metastasis suppressor gene 1 (TMSG1) was a novel tumor metastasis-related gene identified using messenger RNA differential display from non-metastatic human prostate cancer cell variants. The mechanism of LASS2/TMSG1 inhibiting tumor invasion metastasis in breast cancer cells had not been well investigated. In the present study, a full length of 1.2kb LASS2/TMSG1 complementary DNA (cDNA) coding for a protein of 380 amino acids was cloned. PcDNA3 eukaryotic expression plasmids of LASS2/TMSG1 were constructed and transfected into human breast cancer cell line MCF-7 by lipofectin transfection method. And, the biological effects were observed comparing with control groups. As the result, LASS2/TMSG1 inhibited cell growth in vitro by increasing apoptosis and changing cell cycle distribution. Furthermore, the vacuolar ATPase (V-ATPase) activity and extracellular hydrogen ion concentration were significantly decreased and the activity of secreted matrix metalloproteinase-2 (MMP-2) was downregulated in MCF-7 cells overexpressing LASS2/TMSG1 compared with the controls. Therefore, LASS2/TMSG1 may inhibit growth and invasion of breast cancer cell in vitro through decreasing V-ATPase activity and extracellular hydrogen ion concentration and inactivating secreted MMP-2. The findings provided the evidence that the LASS2/TMSG1 gene had tumor growth and invasion suppressor function in human breast cancer cell and may provide a promising target for cancer metastasis diagnosis and therapy.

Open microsurgical tumor excavation and vertebroplasty for metastatic destruction of the second cervical vertebra—outcome in seven cases

Background contextMetastatic osteolytic involvement of the second cervical vertebra (C2) is rare, but usually very painful. Percutaneous vertebroplasty has shown to be effective regarding pain control, but carries the risk of cement leakage. PurposeTo describe an alternative microsurgical procedure suitable for patients suffering from C2 osteolysis who are considered to be high risk with respect to cement leakage. Study designA technical report. Patient sampleIt included seven patients. Outcome measuresThey include the assessment of clinical safety regarding approach- and procedure-related morbidity and radiologic safety regarding extravertebral cement leakage and the assessment of clinical efficacy by monitoring the pain activity using the visual analog scale (VAS). Materials and methodsSeven patients (five men, two women; mean age 70 years) presented with an acute onset of excruciating neck pain (VAS>6) due to osteolytic destruction of the axis vertebra. There was no neurologic deficit and no compression of the spinal cord preoperatively requiring surgical decompression or stabilization in any of the cases. An open treatment strategy via an anterolateral microsurgical approach was performed. Under biplanar fluoroscopic control, the soft tumor tissue was resected out of the vertebral body through a drilled entry in the anterior wall. After the excavation procedure, the resection cavity was filled with minimal pressure with polymethylmethacrylate bone cement. ResultsAll patients suffered from severe spontaneous neck pain (mean VAS 8.1, range 6–9), with head motion-dependent pain exacerbation despite high dose of opiates and fixation of the head with a brace.Mean duration of the operative procedure was 51 minutes. Histologic analysis revealed a diagnosis of cancer metastasis in all cases. On average, 1.9 mL cement was placed within the vertebral body, and no cement leakage was observed in postoperative computed tomography and X-ray controls. All patients experienced immediate pain relief at Day 1 after the procedure (mean VAS 4.0, range 2–6), and a further decrease of pain levels was observed at Week 6 after the completion of radiation therapy (mean VAS 2.0, range 0–5). ConclusionsIn cases of metastatic C2 destruction, tumor excavation via an anterolateral approach and subsequent filling of the resection cavity with bone cement offers a safe and effective alternative to percutaneous approaches.

Nanobiodevice-based single biomolecule analysis, single-cell analysis, and in vivo imaging for cancer diagnosis, cancer theranostics, and iPS cell-based regenerative medicine.

Numerous nanobiodevices have been developed for cancer diagnosis through the analysis of cancer cells and cancer-related biomolecules, cancer gene therapy, and iPS cell-based regenerative medicine. A microchamber array, which is fabricated on a plastic chip, enables to develop a reliable circulating tumor cell (CTC) detection technique for cancer metastasis diagnosis. A nanopillar-array or a nanowire-array on a quartz chip allows ultrafast analysis of DNA and microRNA within 1 s for the molecular diagnosis of cancer. Immunopillar devices, which contain a tremendous amount of antibody-immobilized microparticles inside an immunopillar structure, have realized fast and low invasive "from blood to analysis" type biomarker detection of cancer with a pM detection sensitivity within 5 min. Quantum dots and gene delivery nanodevices are applied to single cancer cell diagnosis, in vivo imaging for iPS cell based regenerative medicine, and theranostic devices for cancer diagnosis and therapy.

DNA Nanospheres with Microfluidics: A Promising Platform for Cancer Diagnosis?

The mortality rate from cancer in the USA reached more than 570,000 in 2012, or equal to more than 1500 Americans each day [1]. More than 90% of cancer deaths result from metastasis [2], hence methods for early-stage cancer detection are vital for the reduction of mortality and they will have a significant medical and social impact. However, according to the Early Detection Research Network of the National Cancer Institute, ‘there are no validated molecular biomarker tests for the early detection of any cancer’ [3], even though a handful of biomarkers have been approved by the US FDA. It is known that, during metastasis, some cancer cells escape from the primary tumor through exfoliation, entering the bloodstream and becoming circulating tumor cells (CTCs) in the peripheral blood stream [2,4]. Some of these CTCs acquire the capability to colonize secondary sites, spreading tumors to distant organs [2,4]. Therefore, CTCs have the potential to serve as important biomarkers for early diagnosis of cancer metastasis [2,4]. An additional benefit is that CTC enumeration is less invasive than biopsy, while providing a quantifiable method for cancer diagnosis and prognosis. However, CTCs are extremely rare; the number of CTCs is in the order of one to 100 cells in 1 ml of blood [4]. For comparison, other cells in 1 ml of blood include approximately 5 × 10 erythrocytes, 7.8 × 10 leukocytes and approximately 3.5 × 10 platelets [5]. Therefore, detection of CTCs in peripheral blood is a needlein-a-haystack challenge. Currently, CellSearch (Veridex LLC, NJ, USA) is the only FDAapproved method, and it relies on antibodycoated magnetic particles for isolation of CTCs from blood [4]. One of its key drawbacks is low capture efficiency. Microfluidics could play an important role in enhancing the capture efficiency of CTCs.

Highly sensitive enumeration of circulating tumor cells in lung cancer patients using a size-based filtration microfluidic chip

Circulating tumor cells (CTCs) in the peripheral blood could serve as a surrogate marker for the diagnosis of cancer metastasis and for therapeutic evaluation. However, the separation and characterization of CTCs is technically challenging owing to the extremely low number of CTCs present. Here we developed a size-based and high-throughput microfluidic chip, which exploits filtration microchannels to isolate the relatively larger CTCs from the rest of the blood constituents. High isolation efficiency of our microfluidic chip was demonstrated with three lung cancer cell lines spiked in blood samples at an optimal flow rate of 0.4mL/h. The average recovery rates of 96%, 95% and 92% were obtained for A549, SK-MES-1, and H446, respectively. To clinically validate the chip, we also employed it to isolate CTCs from 59 lung cancer patients. CTCs were detected in 96.7% of patients with the mean number of 18.6cells/mL, which was significantly higher than normal controls (P<0.05). The work here indicates that the size-based microfluidic platform with the advantage of capturing tumor cells without reliance on cell surface expression markers can provide a novel, inexpensive and effective tool for CTC detection and evaluation of cancer status.

Advances of lab-on-a-chip in isolation, detection and post-processing of circulating tumour cells

Circulating tumour cells (CTCs) are shed by primary tumours and are found in the peripheral blood of patients with metastatic cancers. Recent studies have shown that the number of CTCs corresponds with disease severity and prognosis. Therefore, detection and further functional analysis of CTCs are important for biomedical science, early diagnosis of cancer metastasis and tracking treatment efficacy in cancer patients, especially in point-of-care applications. Over the last few years, there has been an increasing shift towards not only capturing and detecting these rare cells, but also ensuring their viability for post-processing, such as cell culture and genetic analysis. High throughput lab-on-a-chip (LOC) has been fuelled up to process and analyse heterogeneous real patient samples while gaining profound insights for cancer biology. In this review, we highlight how miniaturisation strategies together with nanotechnologies have been used to advance LOC for capturing, separating, enriching and detecting different CTCs efficiently, while meeting the challenges of cell viability, high throughput multiplex or single-cell detection and post-processing. We begin this survey with an introduction to CTC biology, followed by description of the use of various materials, microstructures and nanostructures for design of LOC to achieve miniaturisation, as well as how various CTC capture or separation strategies can enhance cell capture and enrichment efficiencies, purity and viability. The significant progress of various nanotechnologies-based detection techniques to achieve high sensitivities and low detection limits for viable CTCs and/or to enable CTC post-processing are presented and the fundamental insights are also discussed. Finally, the challenges and perspectives of the technologies are enumerated.

Aptamer-Enabled Efficient Isolation of Cancer Cells from Whole Blood Using a Microfluidic Device

Circulating tumor cells (CTC) in the peripheral blood could provide important information for diagnosis of cancer metastasis and monitoring treatment progress. However, CTC are extremely rare in the bloodstream, making their detection and characterization technically challenging. We report here the development of an aptamer-mediated, micropillar-based microfluidic device that is able to efficiently isolate tumor cells from unprocessed whole blood. High-affinity aptamers were used as an alternative to antibodies for cancer cell isolation. The microscope-slide-sized device consists of >59,000 micropillars, which enhanced the probability of the interactions between aptamers and target cancer cells. The device geometry and the flow rate were investigated and optimized by studying their effects on the isolation of target leukemia cells from a cell mixture. The device yielded a capture efficiency of ~95% with purity of ~81% at the optimum flow rate of 600 nL/s. Further, we exploited the device for isolating colorectal tumor cells from unprocessed whole blood; as few as 10 tumor cells were captured from 1 mL of whole blood. We also addressed the question of low throughput of a typical microfluidic device by processing 1 mL of blood within 28 min. In addition, we found that ~93% of the captured cells were viable, making them suitable for subsequent molecular and cellular studies.

Comorbidity as a Mediator of Survival Disparity Between Younger and Older Women Diagnosed With Metastatic Breast Cancer

The presence of comorbidity becomes increasingly important for its prognostic effect on survival in breast cancer patients with advancing age. This study aimed to evaluate the role of comorbidities including hypertension as a mediator of disparity in survival after metastasis diagnosis between younger (≤51 years) and older (>51 years) patients. A total of 553 patients 26-88 years of age with breast cancer metastasis diagnosis from 1 large urban practice were followed between January 1, 1999, and June 30, 2008. Comorbidity variables and survival were analyzed using Cox regression model. To assess comorbidity variables as a mediator of age-survival relationship, 2 approaches have been applied: (1) Baron Kenny approach and (2) alternative assessment to compute the percentage change in the hazard ratios (HRs). The median survival was 40 months, with 265 (47.9%) alive and 288 (52.1%) dead. Older patients had worse survival than younger patients (HR, 1.43; 95% confidence interval [CI], 1.11-1.84). Hypertension was related to survival (HR, 1.45; 95% CI, 1.12-1.89) when age and other covariates were controlled. The effect of age on survival was no longer significant after adjustment for hypertension (HR, 1.26; 95%, CI 0.97-1.65) or hypertension-augmented Charlson comorbidity score (HR, 1.24; 95% CI, 0.95-1.63). Hypertension-augmented Charlson comorbidity score or hypertension was a strong mediator of age-survival relationship among metastatic breast cancer patients, explaining survival disparity between younger and older patients by 44% and 40%, respectively. The study findings suggest that hypertension should be included in the comorbidity information for decision-making support programs.

Factors associated with mortality after breast cancer metastasis

It is generally accepted that patients with breast cancer metastases have very poor survival. Metastatic breast cancer patients can be considered a heterogeneous population with a varied clinical course, which underscores the need for accurate prediction of survival based on prognostic factors. The purpose of the present study was to identify factors related to survival in breast cancer patients after diagnosis with metastatic disease. A total of 557 patients with breast cancer metastasis diagnosis seen at one large urban practice have been followed up between 1 January, 1999 and 30 June, 2010. Demographic, tumor characteristics, clinical factors as predictors of survival were analyzed using log-rank test and Cox regression model. The median survival length was 39 months (range 1-138 months) with 154 (27.7%) alive and 403 (72.3%) dead at the end of follow-up period. This study demonstrated that a history of hypertension, ER/PR status, HER2 status, metastasis-free interval, metastatic location (including brain, bone and liver), and BMI at diagnosis with metastatic breast cancer were the most relevant prognostic factors for survival after metastatic disease diagnosis. Findings of this study may form a foundation for the growing corpus of knowledge explaining the outcome differences in treatment of patients with metastatic breast cancer, potentially helping to create tailored counseling and personalized treatment approaches for this vulnerable group.

Factors associated with mortality after breast cancer metastasis.

174 Background: It is generally accepted that patients with breast cancer metastases have poor survival. Metastatic breast cancer patients can be considered a heterogeneous population with a varied clinical course, which underscores the need for accurate prediction of survival based on prognostic factors. The purpose of the present study was to identify factors related to survival in breast cancer patients after diagnosis with metastatic disease. Methods: A total of 557 patients with breast cancer metastasis diagnosis seen at one large urban practice have been followed up between January 1, 1999 and June 30, 2008. Demographic, tumor characteristics, clinical factors as predictors of survival were analyzed using Cox regression model. Results: The median survival length was 40 months (range 1-114 months) with 269 (48.3%) alive and 288 (51.7%) dead. This study demonstrated that hypertension, estrogen receptor (ER) and/or progesterone receptor (PR) status, human epidermal growth factor receptor-2 (HER2) status, number of metastatic sites, and body mass index (BMI) at diagnosis with metastatic breast cancer were the most relevant prognostic factors for survival after metastasis. Conclusions: Findings of this study may form a foundation for the corpus of knowledge explaining the outcome differences in treatment of patients with metastatic breast cancer, potentially helping to create tailored counseling and personalized treatment approaches for this vulnerable group. [Table: see text]

The effect of delays in treatment for breast cancer metastasis on survival

It is generally accepted that delay in receiving treatment for breast cancer results in adverse outcomes. The purpose of this study was to evaluate the impact of delay in treatment after the diagnosis of metastatic disease on survival measured from metastatic breast cancer diagnosis and from first treatment while controlling for immortal time effect among patients with metastatic breast cancer. A total of 553 patients with breast cancer metastasis diagnosis from one large urban practice have been followed between January 1, 1999 and June 30, 2008. Prognostic factors and outcomes of these patients were analyzed using log-rank test and Cox regression model. Backward stepwise selection of covariates was conducted to assess the association of treatment delay with survival. The median survival was 40 months (range 1-114 months), with 265 (47.9%) women alive and 288 (52.1%) having died at the end of the follow-up period. Treatment delays of more than 12 weeks had impact on poor survival from first treatment than the delays of 4-12 weeks with borderline significance level (HR 1.76, 95% CI 0.99-3.13, P = 0.056) in multivariate analysis, adjusted by BMI, history of hypertension, ER/PR status, HER2 status, number of metastatic sites, and liver metastasis. Moreover, the interval of 12-24 weeks, compared to the interval of 4-12 weeks was associated with greater risk of death from first treatment (HR 2.39, 95% CI 1.19-4.77, P = 0.014). The treatment delay interval of >12 weeks was not related with survival since metastatic breast cancer diagnosis, compared to the 4-12 weeks of treatment delays. This study demonstrated that delays of over 12 weeks in receiving treatment for metastatic breast cancer were related to adverse survival outcomes measured from initiation of first treatment. The findings of this study support targeted efforts to ensure prompt treatment initiation in patients diagnosed with metastatic breast cancer.

Enhancement of the photostability and retention time of indocyanine green in sentinel lymph node mapping by anionic polyelectrolytes

Sentinel lymph node (SLN) biopsy techniques have been widely used in the diagnosis of cancer metastasis because lymph node metastasis is one of the most important prognostic signs. Indocyanine green (ICG) has potential application as a molecular imaging probe for SLN mapping due to its fluorescent properties emitting in the near-infrared (NIR) region, where light transmission through biological tissue is maximized. However, its low photostability in an aqueous solution at the physiological temperature and its rapid diffusion behavior through SLN into the second lymph node have limited its wide use in real clinical fields. In this study, we developed a new NIR imaging contrast system consisting of ICG and poly (γ-glutamic acid) (γ-PGA) polymers for efficient sentinel lymph node mapping. By a combination of clinically used ICG and the biocompatible anionic polyelectrolyte, γ-PGA, the photostabilities of aqueous ICG solutions at room and body temperatures were drastically enhanced. When the ICG/γ-PGA complex was injected subcutaneously into the front paw of a mouse, it entered the lymphatics and migrated to the axillary sentinel lymph node (SLN) within 2 min. Furthermore, the NIR fluorescent signal intensity and retention time of ICG/γ-PGA complex in lymph node were superior to those of ICG only. In addition, a histofluorescentstudy of the SLN resected under NIR imaging revealed that ICG and γ-PGA were co-localized in the lymph node.Taken together, the experimental results on the enhanced photostability and retention time of the ICG/γ-PGA complex provide strong evidence that it has promising potential for improved sentinel lymph node mapping.

A small MRI contrast agent library of gadolinium(III)-encapsulated supramolecular nanoparticles for improved relaxivity and sensitivity

We introduce a new category of nanoparticle-based T1 MRI contrast agents (CAs) by encapsulating paramagnetic chelated gadolinium(III), i.e., Gd3+·DOTA, through supramolecular assembly of molecular building blocks that carry complementary molecular recognition motifs, including adamantane (Ad) and β-cyclodextrin (CD). A small library of Gd3+·DOTA-encapsulated supramolecular nanoparticles (Gd3+·DOTA⊂SNPs) was produced by systematically altering the molecular building block mixing ratios. A broad spectrum of relaxation rates was correlated to the resulting Gd3+·DOTA⊂SNP library. Consequently, an optimal synthetic formulation of Gd3+·DOTA⊂SNPs with an r1 of 17.3 s−1 mM−1 (ca. 4-fold higher than clinical Gd3+ chelated complexes at high field strengths) was identified. T1-weighted imaging of Gd3+·DOTA⊂SNPs exhibits an enhanced sensitivity with a contrast-to-noise ratio (C/N ratio) ca. 3.6 times greater than that observed for free Gd3+·DTPA. A Gd3+·DOTA⊂SNPs solution was injected into foot pads of mice, and MRI was employed to monitor dynamic lymphatic drainage of the Gd3+·DOTA⊂SNPs-based CA. We observe an increase in signal intensity of the brachial lymph node in T1-weighted imaging after injecting Gd3+·DOTA⊂SNPs but not after injecting Gd3+·DTPA. The MRI results are supported by ICP-MS analysis ex vivo. These results show that Gd3+·DOTA⊂SNPs not only exhibits enhanced relaxivity and high sensitivity but also can serve as a potential tool for diagnosis of cancer metastasis.

Imprint cytodiagnosis of lymph node metastasis in resected lung cancer

The cytological examination of lymph node imprints is a rapid and useful method for diagnosis of lymph node metastasis. We applied this technique for the diagnosis of regional lymph node metastasis of resected lung cancer, and compared its diagnostic value with histological examination. The accuracy rate of imprint cytology is 97.3%, and the sensitivity rate is 100%. We conclude that imprint cytology of lymph node for diagnosis of cancer metastasis is highly accurate and simple, and its diagnostic value may be equal to that of routine histological examination.

Intraoperative diagnosis of cancer metastasis in sentinel lymph node of oral cancer patients

Sentinel lymph node (SLN) biopsy in the head and neck region is attracting attention. If intraoperative frozen section and/or cytology of SLN is available, one can select an appropriate patient who must undergo neck dissection in a one-stage procedure. We began intraoperative diagnosis of SLN biopsy in patients who underwent oral cancer surgery in 2003. From August 2003 to December 2006, 44 previously untreated patients were accumulated. All patients underwent SLN biopsy prior to the resection of primary cancer. Intraoperative diagnosis of SLN biopsy was performed by multislice frozen section analysis. Patients with positive SLN underwent immediate neck dissection in the same session. Imprint cytology specimen was prepared at the same time. The results of frozen section analysis and imprint cytology were compared with postoperative pathologic diagnosis of permanent specimens. The sensitivity, specificity, overall accuracy, positive and negative predictive value of intraoperative multislice frozen section analysis in lymph node basis were 90.9%, 100%, 99.1%, 100% and 99.0%, respectively. On the other hand, the indexes of imprint cytology were 27.3%, 99.0%, 92.0%, 75.0% and 92.6%, respectively. All indexes of intraoperative frozen section analysis were superior to imprint cytology. In our experience, multislice frozen section analysis surpasses imprint cytology in intraoperative diagnosis of SLN biopsy.

Tumor-Induced Sentinel Lymph Node Lymphangiogenesis and Increased Lymph Flow Precede Melanoma Metastasis

Lymphangiogenesis is associated with human and murine cancer metastasis, suggesting that lymphatic vessels are important for tumor dissemination. Lymphatic vessel alterations were examined using B16-F10 melanoma cells implanted in syngeneic C57Bl/6 mice, which form tumors metastasizing to draining lymph nodes and subsequently to the lungs. Footpad tumors showed no lymphatic or blood vessel growth; however, the tumor-draining popliteal lymph node featured greatly increased lymphatic sinuses. Lymph node lymphangiogenesis began before melanoma cells reached draining lymph nodes, indicating that primary tumors induce these alterations at a distance. Lymph flow imaging revealed that nanoparticle transit was greatly increased through tumor-draining relative to nondraining lymph nodes. Lymph node lymphatic sinuses and lymph flow were increased in mice implanted with unmarked or with foreign antigen-expressing melanomas, indicating that these effects are not due to foreign antigen expression. However, tumor-derived immune signaling could promote lymph node alterations, as macrophages infiltrated footpad tumors, whereas lymphocytes accumulated in tumor-draining lymph nodes. B lymphocytes are required for lymphangiogenesis and increased lymph flow through tumor-draining lymph nodes, as these alterations were not observed in mice deficient for B cells. Lymph node lymphangiogenesis and increased lymph flow through tumor-draining lymph nodes may actively promote metastasis via the lymphatics.

Sentinel lymph node radiolocalization in clinically negative neck oral cancer

The sentinel node concept has become one of the most interesting topics in the treatment of head and neck cancer. The aim of this article is to report the results of our feasibility study and clinical application of sentinel lymph node (SLN) radiolocalization and biopsy in patients with clinically negative neck oral cancer. Individuals with previously untreated N0 oral cancer participated in the study. The radioactive tracer used was 99m Tc phytate. Lymphoscintigrams were taken in the feasibility study, and fusion images of SPECT and CT were obtained in the clinical SLN biopsy (SLNB) group. In the feasibility study, metastases to SLNs and other nodes were analyzed in permanent specimens. In the clinical application group, we investigated the comparative effectiveness of multi-slice frozen section analysis and imprint cytology for the intraoperative diagnosis of SLNB. Fifteen individuals participated in the feasibility study. Six SLNs in five patients were cancer-positive, and two thirds of the SLNs were micrometastases. The SLN concept was established, and SLNs with the highest to the third highest radioactivity reflected the patients' neck status accurately. Twelve patients participated in the clinical application group of SLNB. Intraoperative diagnosis of the three hottest SLNs correctly predicted the neck status of 10 patients. Three patients underwent modified radical neck dissection on the basis of the intraoperative diagnosis of cancer metastasis to SLNs, whereas neck dissections were spared in patients with no evidence of such metastases. There were two false-negative cases. One involved a failure of the intraoperative diagnosis of SLNB, and the other had cancer-negative SLNs and cancer-positive non-SLNs. Considering intraoperative diagnosis, multi-slice frozen section analysis was found to be superior to imprint cytology in its sensitivity, specificity, and overall accuracy on a lymph node basis. No differences were found in any of these indices of intraoperative SLNB on a patient basis. The fusion images of SPECT and CT proved very useful during intraoperative SLNB. The sentinel node concept was established in the head and neck region. Analyzing the three hottest SLNs suffices to predict a patient's neck status. Multi-slice frozen section analysis was shown to be superior to imprint cytology for detecting micrometastasis to SLN. Intraoperative SLNB based on fusion images of SPECT and CT proved to be an easy, accurate, and reliable method.