Tumor Cells In Sentinel Lymph Node Research Articles

Controversies in Sentinel Lymph Node Biopsy for Gynecologic Malignancies

Sentinel lymph node (SLN) biopsy represents an evolution in the advancement of minimally invasive surgical techniques for gynecologic cancers. Prospective and retrospective studies have consistently shown its accuracy in the detection of lymph node metastases for endometrial and cervical cancers. However, consistent with any emerging surgical technique in the early phases of adoption, new questions have arisen regarding its application and impact. This paper served as a scoping review to identify the key controversies that have arisen in the field of SLN biopsy for endometrial and cervical cancers. Several key controversies were identified, and PubMed, the Cochrane Library (cochranelibrary.com) advanced search function, and the National Comprehensive Cancer Network guidelines were searched for supporting evidence. These included search terms such as "the accuracy of SLN biopsy for high grade endometrial cancer or cervical cancers >2-cm," "cost effectiveness of SLN biopsy for gynecologic cancers," "clinical significance of low volume metastases in endometrial cancer," "morbidity of SLN biopsy for endometrial and cervical cancer," and "impact on cancer survival of SLN biopsy for endometrial and cervical cancer." Studies were selected for review if they included significant numbers of patients, were level I evidence, or were prospective trials. Where this level of evidence failed to exist, seminal observational series that were published in high-quality journals were included. Similar studies were listed and subcategorized and cross-compared, excluding those that included repeated analyses of the same patient populations. The relevant clinical trials or observational studies were clustered and reviewed for each chosen controversy. Adequate evidence supports the accuracy of SLN biopsy in the staging of high-grade endometrial cancer and cervical cancer, and it seems to be a cost-effective strategy for invasive endometrial cancer. Conclusive evidence was lacking with respect to the oncologic outcomes related to SLN biopsy, the impact on patient morbidity, and whether clinicians should treat isolated tumor cells in SLNs with adjuvant therapy. SLN biopsy is an accepted staging strategy for cervical and endometrial cancer surgery; however, controversies remain in how it can be applied with the most safety and efficacy. These ultimately need to be resolved with further clinical trials and observations of larger series of patients.

Sentinel lymph node biopsy in early stage endometrial cancer: a Turkish gynecologic oncology group study (TRSGO-SLN-001)

ObjectiveThe aim of this multicenter study was to evaluate the feasibility of sentinel lymph node (SLN) mapping in clinically uterine confined endometrial cancer.MethodsPatients who underwent primary surgery for endometrial cancer...

Tumor-targeting CuS nanoparticles for multimodal imaging and guided photothermal therapy of lymph node metastasis

Precise diagnosis of lymph node metastasis to guide lymphadenectomy is highly important for gastric cancer therapy in clinics. Though surgical dissection of regional metastatic lymph nodes remains the only way for gastric cancer therapy, the extended dissection may cause unavoidable postoperative risk of complications. It is still lack of effective method enabling the accurate removal of metastatic gastric cancer cells in lymph nodes with minimum injuries to normal tissue. Herein, we report a new fluorescent copper sulfide (CuS) nanoparticle (RGD-CuS-Cy5.5) enabling both non-invasive multimodality imaging and targeting photothermal therapy (PTT) of metastatic gastric cancer cells in lymph nodes. We demonstrate that RGD-CuS-Cy5.5 can easily drain into sentinel lymph nodes (SLN) after injection into primary tumors, and selectively enter into metastatic gastric MNK45 tumor cells via αvβ3 integrin-mediated endocytosis. The resulting strong near-infrared (NIR) fluorescence and computed tomography (CT) contrast in metastatic SLN compared to normal SLN can precisely differentiate SLN metastasis of gastric cancers. Guided by the imaging, localized PTT with RGD-CuS-Cy5.5 is conducted upon irradiation with an 808 nm laser, resulting in complete removal of metastatic gastric tumor cells in SLN without obvious toxicity. Moreover, RGD-CuS-Cy5.5 can also allow for the rapid and non-invasive self-monitoring of PTT efficacy against metastatic SLNs in living mice. This study highlights the potential of using RGD-CuS-Cy5.5 for imaging-guided and targeting PTT of SLN metastasis in vivo, which may be applicable for the metastatic gastric cancer therapy in clinics. Statement of SignificanceRGD-CuS-Cy5.5 nanoparticles possess NIR fluorescence and CT signals for in vivo bimodality imaging of lymph node metastasis. Strong photothermal property under irradiation at 808 nm for efficient PTT. Easy drain into sentinel lymph nodes and selective enter metastatic gastric cancer cells via αvβ3 integrin-mediated endocytosis. Rapid and non-invasive monitoring of therapeutic efficacy against lymph node metastasis.

Can flow cytometry reinvent the sentinel lymph node concept in gastric cancer patients?

BackgroundThe focused sentinel lymph node (SLN) concept we proposed previously relied on real time-quantitative polymerase chain reaction (RT-qPCR) to detect tumor cells, which is too elaborate for intraoperative use. Therefore, we evaluated flow cytometry for intraoperative detection of tumor cells in SLNs. MethodsSixty-five consecutive gastric cancer patients were included. SLN analysis was carried out for a single SLN from each patient, using the molecular methods of RT-qPCR (first 30 patients) and flow cytometry (final 35 patients). All LNs underwent hematoxylin and eosin staining and immunohistochemical examination. ResultsExtraction of the SLN from a high-risk station was an important determinant for accurate prediction of LN metastases. For RT-qPCR, the sensitivity and specificity of detection were 72.7% and 81.8%, respectively, and for flow cytometry, 36.8% and 100%, respectively. When only high-risk SLNs were analyzed and specimens with <10% viability of leukocytes were excluded, the sensitivity and specificity of flow cytometry were 60% and 100%, respectively. Multivariate analysis identified significant predictors for LN metastases as the molecular method of SLN analysis (P = 0.021; 95% confidence interval [CI]: 1.304-24.284) and lymphovascular invasion (P = 0.002; 95% CI: 2.142-28.555). In subgroup analysis of high-risk SLNs, only RT-qPCR was a significant predictor for LN metastases (P = 0.016; 95% CI: 1.581-91.084). ConclusionsFlow cytometry of high-risk SLNs, excluding specimens with low cell viability is a rapid, cost-effective, widely obtainable, and highly specific method for SLN metastases detection although it lacks the necessary sensitivity. Therefore, it cannot be recommended as a stand-alone method for the detection of LN metastases during operations.

Gene promoter hypermethylation is found in sentinel lymph nodes of breast cancer patients, in samples identified as positive by one-step nucleic acid amplification of cytokeratin 19 mRNA.

We analysed the promoter methylation status of five genes, involved in adhesion (EPB41L3, TSLC-1), apoptosis (RASSF1, RASSF2) or angiogenesis (TSP-1), in intraoperative sentinel lymph node (SLN) biopsy samples from patients with breast cancer, that had been processed by the one-step nucleic acid amplification (OSNA) technique. SLN resection is performed to estimate the risk of tumour cells in the clinically negative axilla, to avoid unnecessary axillary lymph node dissection. OSNA is currently one of the eligible molecular methods for detecting tumour cells in SLNs. It is based on the quantitative evaluation of cytokeratin 19 mRNA which allows distinguishing between macrometastasis, micrometastasis and isolated tumour cells, on the basis of the quantity of tumour cells present. There have been no prior studies on the question whether or not samples processed by OSNA can be used for further molecular studies, including epigenetic abnormalities which are some of the most important molecular alterations in breast cancer. Genomic DNA was extracted from samples obtained from 50 patients diagnosed with primary breast cancer. The content of tumour cells in SLNs was evaluated by OSNA, and the promoter methylation status of the selected genes was analysed by methylation-specific PCR. All were found to be hypermethylated to a variable degree, and RASSF1 hypermethylation was significantly associated with macrometastasis, micrometastasis and isolated tumour cells (p=0.002). We show that samples used for OSNA are suitable for molecular studies, including gene promoter methylation. These samples provide a new source of material for the identification of additional biomarkers.

Prognostic significance of axillary dissection in breast cancer patients with micrometastases or isolated tumor cells in sentinel nodes: a nationwide study.

We estimated the impact of axillary lymph node dissection (ALND) on the risk of axillary recurrence (AR) and overall survival (OS) in breast cancer patients with micrometastases or isolated tumor cells (ITC) in sentinel nodes. We used the Danish Breast Cancer Cooperative Group (DBCG) database to identify patients with micrometastases or ITC in sentinel nodes following surgery for primary breast cancer between 2002 and 2008. A Cox proportional hazard regression model was developed to assess the hazard ratios (HR) for AR and OS between patients with and without ALND. We identified 2074 patients, of which 240 did not undergo further axillary surgery. The 5-year cumulated incidence for AR was 1.58 %. No significant difference in AR was seen between patients with and without ALND. The age adjusted HR for AR if ALND was omitted was 1.79 (95 % CI 0.41-7.80, P = 0.44) in patients with micrometastases and 2.21 (95 % CI 0.54-8.95, P = 0.27), in patients with ITC after a median follow-up of 6 years and 3 months. There was no significant difference in overall survival between patients with and without ALND, when adjusting for age, co-morbidity, tumor size, histology type, malignancy grade, lymphovascular invasion, hormone receptor status, adjuvant systemic treatment and radiotherapy, with a HR for death if ALND was omitted of 1.21 (95 % CI 0.86-1.69, P = 0.27) in patients with micrometastases and 0.96 (95 % CI 0.57-1.62, P = 0.89) in patients with ITC after a medium follow-up on 8 and 5 years. In this nationwide study, we found a low risk of AR on 1.58 % and we did not find a significantly increased risk of AR if ALND was omitted in patients with micrometastases or ITC in sentinel nodes. Furthermore, no significant difference in overall survival was seen between patients with and without ALND when adjusting for adjuvant treatment.

Robust and validated models to predict high risk of non-sentinel node metastases in breast cancer patients with micrometastases or isolated tumor cells in the sentinel node

Background. Benefit from axillary lymph node dissection in sentinel node positive breast cancer patients is under debate. Based on data from 1820 Danish breast cancer patients operated in 2002–2008, we have developed two models to predict high risk of non-sentinel node metastases when micrometastases or isolated tumor cells are found in sentinel node. The aim of this study was to validate these models in an independent Danish dataset. Material and methods. We included 720 breast cancer patients with micrometastases and 180 with isolated tumor cells in sentinel node operated in 2009–2010 from the Danish Breast Cancer Cooperative Group database. Accuracy of the models was tested in this cohort by calculating area under the receiver operating characteristic curve (AUC) as well as sensitivity and specificity. Results. AUC for the model for patients with micrometastases was comparable to AUC in the original cohort: 0.63 and 0.64, respectively. The sensitivity and specificity for predicting risk of non-sentinel node metastases over 30% was 0.36 and 0.81, respectively, in the validation cohort. AUC for the model for patients with isolated tumor cells decreased from 0.73 in the original cohort to 0.60 in the validation cohort. When dividing patients with isolated tumor cells into high and low risk of non-sentinel node metastases according to number of risk factors present, 37% in the high-risk group had non-sentinel node metastases. Specificity and sensitivity was 0.48 and 0.88, respectively, in the validation cohort when using this cut-point. Conclusion. In this independent dataset, the model for patients with micrometastases was robust with accuracy similar to the original cohort, while the model for patients with isolated tumor cells was less accurate. The models may be used to identify patients where axillary lymph node dissection should still be considered.

16 Prognostic Significance of Axillary Lymph Node Dissection in Breast Cancer Patients with Micrometastases or Isolated Tumor Cells in Sentinel Nodes – a Nationwide Study

We estimated the impact of axillary lymph node dissection (ALND) on the risk of axillary recurrence (AR) and overall survival (OS) in breast cancer patients with micrometastases or isolated tumor cells (ITC) in sentinel nodes. We used the Danish Breast Cancer Cooperative Group (DBCG) database to identify patients with micrometastases or ITC in sentinel nodes following surgery for primary breast cancer between 2002 and 2008. A Cox proportional hazard regression model was developed to assess the hazard ratios (HR) for AR and OS between patients with and without ALND. We identified 2074 patients, of which 240 did not undergo further axillary surgery. The 5-year cumulated incidence for AR was 1.58 %. No significant difference in AR was seen between patients with and without ALND. The age adjusted HR for AR if ALND was omitted was 1.79 (95 % CI 0.41–7.80, P = 0.44) in patients with micrometastases and 2.21 (95 % CI 0.54–8.95, P = 0.27), in patients with ITC after a median follow-up of 6 years and 3 months. There was no significant difference in overall survival between patients with and without ALND, when adjusting for age, co-morbidity, tumor size, histology type, malignancy grade, lymphovascular invasion, hormone receptor status, adjuvant systemic treatment and radiotherapy, with a HR for death if ALND was omitted of 1.21 (95 % CI 0.86–1.69, P = 0.27) in patients with micrometastases and 0.96 (95 % CI 0.57–1.62, P = 0.89) in patients with ITC after a medium follow-up on 8 and 5 years. In this nationwide study, we found a low risk of AR on 1.58 % and we did not find a significantly increased risk of AR if ALND was omitted in patients with micrometastases or ITC in sentinel nodes. Furthermore, no significant difference in overall survival was seen between patients with and without ALND when adjusting for adjuvant treatment.

Impact of CD44+CD24- cells on non-sentinel axillary lymph node metastases in sentinel node-positive breast cancer

Although complete axillary lymph node dissection (ALND) is the standard for evaluating axillary status after the identification of a positive sentinel lymph node (SLN) in breast cancer; approximately 40-60% of SLN-positive patients have negative non-SLN. In this study, to explore putative breast cancer stem cells with CD44+CD24- in the SLN, we retrospectively analyzed the expression of CD44+CD24- on metastatic tumor cells within SLNs as a predictive factor for positive non-SLNs (NSLNs). We tested 271 patients for SLNs using serial sectioning with cytokeratin immunohistochemistry (IHC) and hematoxylin-eosin staining and identified 67 patients who had a positive SLN biopsy and complete ALND. CD44 and CD24 expression was detected using double-staining IHC. Twenty-eight (41.8%) out of 67 patients had positive NSLN metastases. Seven positive SLNs with micrometastases were not available for the evaluation of CD24 and CD44 expression. Out of the remaining 60patients, 19(31.7%), 44(73.83%) and 37(61.7%) patients had CD24+, CD44+ and CD44+CD24- metastatic tumor cells in SLNs, respectively. Positive NSLN metastasis was significantly associated with the primary tumor size (P=0.004), CD24- expression (P=0.04), CD44+ expression (P=0.01) and CD44+CD24- expression (P=0.02). This report provides the first evidence of the existence of a putative stem-like phenotype within the SLN, which is significantly associated with positive NSLN in early breast cancer patients.

Pathologic Evaluation of Axillary Dissection Specimens Following Unexpected Identification of Tumor Within Sentinel Lymph Nodes

Axillary lymph node dissection has been the standard of care after identification of a positive sentinel lymph node for breast cancer patients. To determine the likelihood of non-sentinel lymph node involvement for patients with negative sentinel node by frozen section, who are subsequently found to have tumor cells in the sentinel node by permanent section levels and/or cytokeratin immunohistochemistry. One hundred three patients with invasive breast cancer exhibiting negative frozen section evaluation of their sentinel node, but later found to have isolated tumor cells (n = 46), micrometastasis (n = 46), or metastases (n = 11) in their sentinel node by permanent sections or immunohistochemistry, were enrolled in this prospective cohort study and underwent completion axillary dissection. Six of 46 patients (13%) with isolated tumor cells in their sentinel node, 15 of 46 patients (33%) with micrometastasis in their sentinel node, and 2 of 11 patients (18%) with metastasis in their sentinel node had additional findings in the nonsentinel nodes. These findings resulted in a pathologic stage change in 2 patients. Predictors of positive nonsentinel nodes were 2 or more positive sentinel nodes (P = .002), sentinel nodes with micrometastasis versus isolated tumor cells (P = .03), and those with angiolymphatic invasion (P = .04). Our findings lend support to axillary node dissection for patients with micrometastasis or metastasis in their sentinel nodes. However, studies with clinical follow-up are needed to determine whether axillary node dissection is necessary for patients with isolated tumor cells in sentinel nodes.

Intraoperative Imprint Cytology Examination of Sentinel Lymph Nodes After Neoadjuvant Chemotherapy in Breast Cancer Patients

Intraoperative imprint cytology (IC) is one of several accurate, proven methods to detect tumor cells in sentinel lymph nodes (SLN) from patients with operable breast cancer. In patients treated with neoadjuvant chemotherapy (NAC), studies have demonstrated the feasibility and accuracy of SLN biopsy procedure. We evaluated the validity of IC for SLN testing in patients after NAC. Patients with infiltrating breast carcinoma receiving NAC (n = 132) were studied prospectively. At surgery, SLN biopsy followed by axillary lymph node dissection was performed. SLN were evaluated using IC in 80 of 132 patients (60%). The results of IC in the adjuvant setting (100 patients) were used for comparison. SLN metastases were correctly identified using IC in 58 of 80 (72%) patients. False negative results were observed in 21 patients. The sensitivity of IC testing was 38.2% and specificity 97.8%. The positive and negative predictive values (PPV and NPV) were 92.9% and 68.2%, respectively. In univariate analysis and multivariate logistic regression analysis, patients with micrometastases or isolated tumor cells in SLN have 2.3 times higher risk of a false negative IC result than patients with macrometastases in SLN (P = .00021; relative risk [RR] = 2.3; 95% confidence interval, 1.37-3.85). The non-NAC group, which contained fewer micrometastatic cases, showed better sensitivity (47.4%) and NPV (88.9%). NAC does not seem to influence the accuracy and sensitivity of IC. Variations in sensitivity are related to the proportion of cases with micrometastases and ITC, as it was also shown in chemonaive patients.

The Impact of CD44+CD24- Cells on Non-Sentinel Axillary Lymph Node Metastases in Sentinel Node-Positive Breast Cancer.

Abstract Background: Although completion axillary lymph node dissection (CALND) is the standard for evaluating axillary status after identification of a positive sentinel lymph node (SLN) in breast cancer, approximately 30-60% of SLN-positive patients have negative non-SLN. It has been recently demonstrated that the putative breast cancer stem cells with CD44+CD24low/- phenotype are associated with self-renewal and tumorigenic potential. In this study, in order to explore such putative breast cancer stem cells in the SLN, we intended to retrospectively analyze expression of CD44+CD24low/- on metastatic tumor cells within SLN as a predictive factor for positive non-SLN (NSLN).Methods: We tested 271 patients for the SLNs by using serial sectioning and cytokeratin immunohistochemistry (IHC) and hematoxylin-eosin, and identified 67 patients who had a positive SLN biopsy and CALND. CD44 and CD24 expression was detected using IHC. Immunostaining for CD44 and CD24 interpreted as positive if at least 1% of tumor cells show distinct membranous staining. Univariate logistic regression techniques were used to assess significant associations.Results: Twenty-seven patients out of 67 patients (40%) had positive NSLN metastases. Six positive SLNs with micrometastases were not available for evaluation of CD24 and CD44 expression. Out of the 61 patients, 22 (36%), 47 (77%) and 28 (62%) patients had CD24-positive, CD44-positive and CD44+/CD24low/- metastatic tumor cells in SLN, respectively. Positive NSLN metastase was significantly associated with primary tumor size (P=0.004), negative CD24 expression (P=0.04), positive CD44 expression (P=0.04) and CD44+/CD24low/- (P=0.03).Conclusion: This is the first evidence for the existence of the putative stem-like phenotype within the SLN, which is significantly associated with positive NSLN, in early breast cancer patients. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1160.

Influence of Isolated Tumor Cells in Sentinel Nodes on Outcome in Small, Node-Negative (pT1N0M0) Breast Cancer

To evaluate the prognostic significance of isolated tumor cells found on sentinel node biopsy. The study is based on a prospectively followed up cohort of 1,865 consecutive patients diagnosed with pT1 (tumor size <or=20 mm) breast cancer in one university breast unit between February 2001 and August 2005. Of the 1,390 patients who received no neoadjuvant therapy and who underwent sentinel node biopsy, 63 had isolated tumor cells in the sentinel nodes (stage pT1N0i + M0, verified by axillary node dissection) and 868 did not (pT1N0i - M0). Median follow-up time was 55 months. Patients with pN0i+ disease received systemic adjuvant therapies more often than those with pN0i- disease (87 versus 51%; P < 0.0001). There was no significant difference between the groups in terms of 5-year recurrence-free survival (90.3 versus 93.2%, respectively; P = 0.32) or overall survival, but patients with pN0i+ cancer had less favorable 5-year breast-cancer-specific survival (95.2 versus 98.4%; P = 0.035) than those with pN0i- cancer, and they were more frequently diagnosed with distant metastases from breast cancer during the first 5 years of follow-up (8.1 versus 1.9%, respectively; P = 0.001). Some conventional prognostic factors, such as histological grade, steroid hormone receptor status, and cell proliferation rate, were more strongly associated with outcome than was pN0i status. The findings suggest that presence of isolated tumor cells in the sentinel nodes is an adverse prognostic factor in early breast cancer, but its prognostic significance in association with standard factors may be limited.

5009 Influence of isolated tumor cells in sentinel nodes on outcome in early pT1N0M0 breast cancer

5009 Influence of isolated tumor cells in sentinel nodes on outcome in early pT1N0M0 breast cancer

Lymphatic mapping and sentinel lymph node detection in women with cervical cancer

Lymphatic mapping and sentinel node detection have been applied to almost every solid tumor and sentinel node status have become part of the American Joint Commission on Cancer (AJCC) staging criteria in both breast cancer and malignant melanoma. As the presence of metastatic disease in lymph nodes is the most important prognostic factor on survival in women with cervical cancer, the ability to reliably detect sentinel nodes might triage women to adjuvant radiotherapy without the need for full lymphadenectomies and their associated morbidity. To date, multiple international investigators have performed single institution investigations with promising results. Overall, 831 women have been undergoing lymphatic mapping and sentinel node detection as part of their cervical cancer therapy as reported in the literature. Combining results from all these studies, a sentinel node was identified in 90% of cases with an overall sensitivity of detecting metastatic disease of 92% with an 8% false negative rate. The overall negative predictive value was over 97%. There remain controversies in moving forward with accepting sentinel node biopsy as the standard in treating women cervical cancer including 1) determining an acceptable false-negative rate, 2) establishing the importance of micrometastatic disease or isolated tumor cells in sentinel nodes, and 3) discovering the minimum number of cases a surgeon needs to become proficient in mapping techniques. Large, multi-institutional studies in both Europe and the United States are nearing completion and their results should help guide the future direction for sentinel node technologies in the treatment of cervical cancer.

Sentinel lymph node as a target of molecular diagnosis of lymphatic micrometastasis and local immunoresponse to malignant cells

The sentinel lymph node (SLN) is defined as the lymph node(s) first receiving lymphatic drainage from the site of the primary tumor. The histopathological status of SLN is one of the most significant predictors of recurrence and overall survival for most clinical stage I/II solid tumors. Recent progress in molecular techniques has demonstrated the presence of micrometastatic tumor cells in SLN. There is now a growing body of data to support the clinical relevance of SLN micrometastasis in a variety of solid tumors. Increasing the sensitivity of occult tumor cell detection in the SLN, using molecular-based analysis, should enable a more accurate understanding of the clinical significance of various patterns of micrometastatic nodal disease. The establishment of metastasis to SLN might not be simply reflected by the flow dynamics of lymphatic fluid that drains from the primary site to the SLN, and the transportation of viable cancer cells. Recent studies have demonstrated that primary tumors can actively induce lymphangiogenesis and promote SLN metastasis. Moreover chemokine receptors in tumor cells may facilitate organ-specific tumor metastasis in many human cancers and some experimental models. In contrast, recent clinical and preclinical studies regard SLN as the first lymphoid organ to respond to tumor antigenic stimulation. SLN dramatically show morphological, phenotypical and functional changes that indicate immune suppression by tumor cells. The immune suppression in SLN results in failure of prevention or eradication of tumor metastasis. The mechanism of immunomodulation remains unclear; however, several regulatory molecules produced by tumor cells and tumor-associated macrophages or lymphocytes are likely to be responsible for inducing the immune suppression in SLN. Further studies may develop a novel immunotherapy that overcomes tumor-induced immune suppression and can prevent or eradicate SLN metastasis.

The value of combined gamma probe and blue dye method for the detection of sentinel lymph nodes in breast carcinoma: a single institution experience

Background: Sentinel lymph node (SLN) biopsy has become a widely used method for a subset for invasive breast carcinoma (IBC) but optimal detection method is controversial. We analyzed combined gamma-probe and blue dye technique for SLN detection in our patients. Patients and methods: 94 patients (34–86yrs, median 62yrs) with IBC <3cm, preoperatively diagnosed by CoreNeedleBiopsy and cN-0 were included. Lymphatic mapping was performed by subareolar or peritumoral injection of 99 mTc-labelled albumin combined with patent-blue V followed by gamma-probe detection and blue-dye visualisation. SLN was defined by either blue-dye staining or by gamma-probe activity. All SLN were examined by frozen section and subsequently totally processed including serial sections and cytokeratin-immunohistochemistry. Results: In 14 cases (=15%) the SLN mapping was discrepant between the 2 methods, in 2 cases the SLN was detected by blue dye only, whereas in 3 cases the SLN were just detected by gamma-probe, in 9 cases a variety of the number of hot and blue SLN existed. Tumour-cells in SLN were found in 34 (=37%) of 91 cases, of which 14 (=41% of positive SLN) were detected by immunohistochemistry. 7 out of the 14 discrepant cases harboured metastases, of which 2 were only detected by blue dye (gamma-probe negative cases), in 3 cases the metastases were located in a SLN that was only gamma probe positive, in 2 further cases the metastases were detected by both methods but further SLN were only gamma probe positive. Conclusions: There is evidence that the single use of either gamma-probe or blue dye may lead to a missing of tumour cells in SLN.

Isolated Tumor Cells in the Sentinel Node Affect Long-Term Prognosis of Patients with Melanoma

The clinical significance of isolated tumor cells (ITCs) in the melanoma-draining sentinel nodes (SNs) is unclear. Records of patients who underwent SN biopsy (SNB) for stage I/II melanoma at our institute between 1991 and 2003 were reviewed to identify patients whose SNs were tumor-free or contained only ITC (<or=0.2 mm). Tumor-positive SNs were reevaluated by the study pathologist to confirm the diagnosis and microstage the SN. Characteristics of the primary melanoma, tumor status of regional lymph nodes, and other prognostic variables were recorded. Melanoma-specific survival (MSS) rates were compared by the log-rank test. Of 1,382 patients who underwent SNB, 1,168 (85%) had tumor-free SNs; among the 214 remaining patients with tumor-positive SNs, 57 had metastases limited to ITC. Completion lymphadenectomy (CLND) was performed in 52 of 57 patients: six (12%) had metastases in nonsentinel nodes (NSNs). At a median follow-up of 57 months, 5-year and 10-year MSS was significantly higher (P = .02) for the 1168 patients with tumor-negative SNs (94 +/- 1% and 87 +/- 2%, respectively) than the 57 patients with ITC-positive SNs (89 +/- 4% and 80 +/- 7%, respectively). Multivariate analysis identified ITC (P = .002), Breslow's thickness (P < .0001), ulceration (P < .0001), and primary site (P = .04) as significant for MSS. Patients with ITC in SNs have a significantly higher risk of melanoma-specific death than those with tumor-negative SNs. The 12% incidence of nonsentinel node metastasis is similar to rates reported for patients with more extensive SN involvement. Patients with ITC should be considered for CLND.

HPV16‐E6 mRNA is superior to cytokeratin 19 mRNA as a molecular marker for the detection of disseminated tumour cells in sentinel lymph nodes of patients with cervical cancer by quantitative reverse‐transcription PCR

About 10-15% of patients with cervical cancer suffer from recurrence despite histologically negative lymph nodes (pN0). Occult micrometastases or small tumour cell clusters may contribute to disease outcome. The aim of this study was to compare at the RNA level 2 known tumour-associated genes, HPV16-E6 and cytokeratin 19 (CK19), as molecular markers for the detection of disseminated tumour cells. Real-time reverse transcription PCR technology was used to quantify gene expression in histologically positive and negative sentinel lymph nodes (SLN) from 70 patients with cervical cancer. Lymph nodes from noncancer patients were used as controls. Calculated copy numbers were normalised to the geometric average of the most stable housekeeping genes. We observed a good correlation (R = 0.915) between the expression of both markers in SLN with histologically confirmed metastases. However, marker gene expression differed considerably in histologically negative nodes: CK19 transcripts were detected in 90 of 112 SLN (80.4%), whereas only 38 nodes (33.9%) were positive for HPV16 E6 mRNA. In particular, 62 of 74 SLN, which were negative by histology, and HPV16 E6 mRNA expressed CK19 mRNA. Moreover, 8 of 10 lymph nodes from noncancer patients expressed CK19 mRNA. Systematic errors due to RNA degradation or incomplete cDNA could be ruled out. It is concluded that HPV16 E6 mRNA is more specific and more sensitive for the detection of tumour cells in SLN than CK19 mRNA. The specificity of CK19 is limited because of low level expression in uninvolved pelvic lymph nodes.

Combined application of RT-PCR and immunohistochemistry on paraffin embedded sentinel lymph nodes of prostate cancer patients

The detection of tumor cells in the sentinel lymph node (SLN) is of great importance for the prognosis of cancer patients. At present, immunohistochemistry and RT-PCR for tumor marker expression are the most sensitive techniques available for this analysis. However, so far, most RT-PCR-based analyses of SLNs have been performed on fresh material, excluding a direct comparison with the (immuno)histologic results. In our view, this does not entirely aid routine diagnosis. We established an efficient method for RNA extraction and RT-PCR from paraffin sections of SLNs from prostate cancer patients and compared the results with the (immuno)histologic data of adjacent sections. Amplifiable RNA was obtained from 133 SLNs of 68 prostate cancer patients. Correlation of PSA-specific RT-PCR with (immuno)histologic findings showed a positive and negative predictive value of 83% and 100%, respectively, for the prostate cancer patients investigated. Four of 12 patients with biochemical relapse, but without (immuno)histologically detectable tumor cells were RT-PCR-positive for PSA. We found that single sections of paraffin-embedded SLNs are suitable for routinely performed RT-PCR. Combined with (immuno)histology, PSA-specific RT-PCR is a revealing supplementary technique for the detection of tumor cells in SLNs of prostate cancer patients.