Molecular analysis of melanoma-induced sentinel lymph node immune dysfunction

Introduction  

Sentinel lymph nodes (SLNs) of melanoma patients show evidence of tumor-induced immune dysfunction. Our previous works have shown that IL-10 and IFNγ co-regulate indoleamine-2,3-dioxygenase (IDO)-expressing immunosuppressive dendritic cells (DCs) in melanoma SLNs. The goal of this study is to examine the relationship between melanoma SLN tumor burden and the degree of SLN immune dysfunction as a model to study tumor-induced immune dysfunction. We hypothesize that SLN tumor burden correlates with the degree of SLN immune dysfunction.     

Imaging tumor-induced sentinel lymph node lymphangiogenesis with LyP-1 peptide

Lymphangiogenesis in tumor-draining lymph nodes (LNs) starts before the onset of metastasis and is associated with metastasis to distant LNs and organs. In this study, we aimed to visualize tumor-induced lymphangiogenesis with a tumor lymphatics-specific peptide LyP-1. The LyP-1 peptide was labeled with a near-infrared fluorophore (Cy5.5) for optical imaging. At days 3, 7, 14 and 21 after subcutaneous 4T1 tumor inoculation, Cy5.5-LyP-1 was administered through the middle phalanges of the upper extremities of the tumor-bearing mice. At 45?min and 24?h postinjection, brachial LN fluorescence imaging was performed. Ex vivo fluorescence images were acquired for quantitative analysis of the fluorescence intensity. Tumor-induced lymphangiogenesis was confirmed by LYVE-1 immunostaining and increased size of tumor side brachial LNs. Cy5.5-LyP-1 staining in LNs co-localized with LYVE-1, suggesting lymphatics-specific binding of LyP-1 peptide. The brachial LNs were clearly visualized by optical imaging at both time points. The tumor side LNs showed significantly higher fluorescence intensities than the contralateral brachial LNs at days 7, 14, and 21, but not day 3 after tumor inoculation. At day 21 after tumor inoculation, the average signal of tumor-draining LNs was 78.0?±?2.44, 24.3?±?5.43, 25.6?±?0.25 (×10³?photon/cm²/s) using Cy5.5-LyP-1, Cy5.5-LyP-1 with blocking, and Cy5.5 only, respectively. Tumor-draining brachial LNs showed extensive growth of lymphatic sinuses throughout the cortex and medulla. Use of LyP-1 based imaging probes with optical imaging offers a useful tool for the study of tumor-induced lymphangiogenesis. LyP-1 may serve as a marker of lymphangiogenesis useful in detecting “high risk” LNs before tumor metastasis and after micro-metastasis, as well as for screening potential anti-lymphatic therapies.     

Preoperative lymphoscintigraphy guided sentinel lymph node biopsy in malignant melanoma

The authors present preliminary experience with preoperative sentinel lymph node biopsy carried out with lymphoscintigraphy in patients with malignant melanoma. PATIENTS AND METHODS: In the present study patients operated for primary cutaneous malignant melanoma of moderate and high severity were included. On the day of surgery isotope labelled colloid was injected intradermally around the tumor to indicate the lymphatics and to obtain basic information about the localization of the sentinel lymph node(s).During surgery the lymph node(s) previously visualized by the injection of patent-blue staining were detected with the aid of a gamma probe. Simultaneously, the excision of the primary tumor was extended. Histologically verified metastasis in the surgically removed lymph node(s) necessitated block dissection possibly within two weeks. RESULTS: The distribution of patients (19) according to tumor localisation: 2 - upper extremities; 9 - lower extremities; 2 - sacral region; 6 - trunk. Tumor thickness ranged from <1.5 mm (6 patients) to 1.5-3 mm (5 patients) and to >3 mm (8 patients). In two cases the identification of the lymph node has failed. Positive sentinel ymph nodes were detected in two patients. It is noteworthy that with one patient the sentinel lymph node was not regional but intransit. This study was aimed at the development of a suitable method. Further on we wish to try it in prospective randomized studies.     

Present status of sentinel lymph node biopsy in cervical cancer

Cervical cancer is the fourth most common cancer in women, and seventh overall. This disease represents a medical, economic and social burden. In early FIGO stage patients (IA, IB1 and IIA1), nodal involvement is the most important prognostic factor. Imaging evaluation of nodal metastasis is of limited value. In order to determine lymph node involvement, allow loco-regional control of the disease, define the need for adjuvant radiotherapy and improve survival, standard surgery for early disease is radical hysterectomy with systematic pelvic lymphadenectomy. However, this surgical treatment has risks and complications: longer operative time, larger blood loss, neurovascular or ureteral injury, lower-limb lymphedema, symptomatic lymphocysts, hydronephrosis. A method that allows to define the presence of regional metastasis with less morbidity and equal or greater precision is particularly relevant. The use of the sentinel lymph node biopsy is intended to reach that purpose. The present study reviews recent literature on the role of sentinel lymph node biopsy in cervical cancer, analyzing its indications and contraindications, injection and detection techniques, tracers used, surgical and pathological approaches and its applicability in up-to-date clinical practice.     

Survival benefit of sentinel lymph node biopsy in Asian melanoma patients

Sentinel lymph node biopsy (SLNB) provides important prognostic information for early-stage melanomas. However, statistics regarding the survival comparison between SLNB and nodal observation in Asia, where acral lentiginous melanoma (ALM) predominates, are limited. This study aimed to identify if SLNB offered survival benefits over nodal observation in early-stage melanomas in Taiwan. The retrospective study included 227 patients who met the SLNB criteria according to the National Comprehensive Cancer Network guidelines and were treated at National Taiwan University Hospital from June 1997 to June 2021. Survival analysis was performed using Kaplan–Meier curves and Cox proportional hazards regression models. Of the study population, ALM accounted for 73.1%; 161 patients (70.9%) underwent SLNB and 66 patients (29.1%) were under nodal observation. Multivariate analysis showed significantly improved melanoma-specific survival (hazard ratio [HR], 0.6; p = .02) in the SLNB group. Among those who underwent completion lymph node dissection (CLND), the non-sentinel node positivity rate was 44.4%. Immediate CLND resulted in significantly longer melanoma-specific survival and distant-metastasis-free survival (DMFS) compared to nodal observation. (HR, 0.2; p = .01 for melanoma-specific survival. HR, 0.3; p = .046 for DMFS). In conclusion, SLNB may provide survival benefits of cutaneous melanoma over nodal observation in the Taiwanese population.     

Near-infrared fluorescent type II quantum dots for sentinel lymph node mapping

The use of near-infrared or infrared photons is a promising approach for biomedical imaging in living tissue. This technology often requires exogenous contrast agents with combinations of hydrodynamic diameter, absorption, quantum yield and stability that are not possible with conventional organic fluorophores. Here we show that the fluorescence emission of type II quantum dots can be tuned into the near infrared while preserving absorption cross-section, and that a polydentate phosphine coating renders them soluble, disperse and stable in serum. We then demonstrate that these quantum dots allow a major cancer surgery, sentinel lymph node mapping, to be performed in large animals under complete image guidance. Injection of only 400 pmol of near-infrared quantum dots permits sentinel lymph nodes 1 cm deep to be imaged easily in real time using excitation fluence rates of only 5 mW/cm(2). Taken together, the chemical, optical and in vivo data presented in this study demonstrate the potential of near-infrared quantum dots for biomedical imaging.     

Pan and sentinel lymph node visualization using a near-infrared fluorescent probe

A number of different types of agents have been employed to aid in the visualization of lymph nodes, particularly the sentinel lymph node, and to decrease the tissue destruction associated with the diagnosis of nodal metastases. The current study was performed to see if a novel macromolecular near-infrared fluorescent (NIRF) probe could be used to visualize lymph nodes after intravenous administration (pan-node visualization) or subcutaneous administration (sentinel node visualization), and serve as method for guiding dissection with interventional radiologic and surgical procedures. Cy5.5-PGC, the near-infrared dye Cy5.5 coupled to a protected graft copolymer (PGC), was injected (i.v. or s.c.) into nude mice. Twenty-four hours later white light and NIRF images were obtained on (i) the live animal, (ii) a partially dissected animal, and (iii) tissue specimens. With Cy5.5-PGC administered intravenously, axillary nodes were visualized from outside a living mouse. With partial dissection, iliac and aortic nodes were visible as concentrated foci of high-intensity NIRF signals. With subcutaneous injection in the front extremity, axillary and brachial nodes draining the injection site were easily visualized. NIRF imaging provides a nonradioactive method of visualizing lymph nodes through layers of tissue that can be employed with intravenous or subcutaneous injection.     

Tumor-derived TGFbeta-1 induces dendritic cell apoptosis in the sentinel lymph node

Lymphatic flux from a primary tumor initially flows into a tumor-draining lymph node (LN), the so-called sentinel LN (SLN). Carried by the lymph fluid are a variety of mediators produced by the tumor that can influence immune responses within the SLN, making it a good model with which to investigate tumor-related immunology. For instance, dendritic cell (DC) numbers are reduced in SLNs from melanoma and breast cancer patients. In the present study, we investigated the mechanism by which DC numbers were reduced within SLNs from patients with non-small cell lung cancer. We found that the incidence of apoptosis among DCs was higher in SLNs than in non-SLNs, as were levels of TGFbeta-1. In contrast, levels of TGFbeta-1 mRNA did not differ between SLNs and non-SLNs, but were 30 times higher in tumors than in either LN type. In vitro, incubation for 2 days with TGFbeta-1 induced apoptosis among both cultured DCs and DCs acutely isolated from normal thoracic LNs, effects that were blocked by the TGFbeta-1 inhibitor DAN/Fc chimera. Taken together, these results suggest that tumor-derived TGFbeta-1 induces immunosuppression within SLNs before the movement of tumor cells into the SLNs, thereby facilitating metastasis within those nodes.     

Implications of axillary sentinel lymph node biopsy in immediate autologous breast reconstruction

For patients with invasive breast cancer, if the results of an axillary sentinel node biopsy are determined to be positive after permanent pathologic examination, the current recommendation is to perform a complete axillary node dissection. Subsequent axillary surgery may compromise the blood supply to an immediate autologous breast reconstruction. The purpose of this study was to determine which clinicopathologic factors in clinically node-negative breast cancer patients may be associated with an increased risk of positive axillary nodes. Identification of these factors will allow surgeons to modify their approach to immediate autologous breast reconstruction in these high-risk patients. The relationship between presenting clinicopathologic characteristics and the incidence of axillary metastases was analyzed by chi-square test and multivariate analysis in 167 patients with invasive breast cancer and a clinically negative axilla who underwent modified radical mastectomy with an immediate free transverse rectus abdominis musculocutaneous (TRAM) flap reconstruction. Axillary nodal metastases were found in 35 percent of clinically node-negative breast cancer patients. Multivariate analysis showed that patient age of 50 years or younger (p = 0.019), T2 tumor stage or greater (p = 0.031), and presence of lymphovascular invasion on the initial biopsy specimen (p < 0.001) were independent predictors of axillary metastases in clinically node-negative patients. Based on these results, the authors propose an algorithm for decision making in clinically node-negative breast cancer patients who desire autologous breast reconstruction and sentinel lymph node biopsy. Options for immediate autologous breast reconstruction in patients undergoing mastectomy and axillary sentinel lymph node biopsy that may minimize the risk of vascular damage on reoperation include the use of the internal mammary artery and vein as recipient vessels for a free TRAM flap or a pedicled TRAM flap. If an axillary-based blood supply is used, the authors are considering the use of cadaveric dermis to isolate the pedicle of the flap away from the remaining axillary contents. New developments in breast cancer diagnosis and treatment necessitate a team approach, with increased communication between the breast surgeon and the plastic surgeon in planning surgery for these patients.     

Organic alternatives to quantum dots for intraoperative near-infrared fluorescent sentinel lymph node mapping

Intraoperative near-infrared (NIR) fluorescence imaging provides the surgeon with real-time image guidance during cancer and other surgeries. We have previously reported the use of NIR fluorescent quantum dots (QDs) for sentinel lymph node (SLN) mapping. However, because of concerns over potential toxicity, organic alternatives to QDs will be required for initial clinical studies. We describe a family of 800 nm organic heptamethine indocyanine-based contrast agents for SLN mapping spanning a spectrum from 775 Da small molecules to 7 MDa nanocolloids. We provide a detailed characterization of the optical and physical properties of these contrast agents and discuss the advantages and disadvantages of each. We present robust methods for the covalent conjugation, purification, and characterization of proteins with tetra-sulfonated heptamethine indocyanines, including mass spectroscopic site mapping of highly substituted molecules. One contrast agent, NIR fluorescent human serum albumin (HSA800), emerged as the molecule with the best overall performance with respect to entry to lymphatics, flow to the SLN, retention in the SLN, fluorescence yield and reproducibility. This preclinical study, performed on large animals approaching the size of humans, should serve as a foundation for future clinical studies.     

Clinical relevance of novel imaging technologies for sentinel lymph node identification and staging

The sentinel lymph node (SLN) concept has become a standard of care for patients with breast cancer and melanoma, yet its clinical application to other cancer types has been somewhat limited. This is mainly due to the reduced accuracy of conventional SLN mapping techniques (using blue dye and/or radiocolloids as lymphatic tracers) in cancer types where lymphatic drainage is more complex, and SLNs are within close proximity to other nodes or the tumour site. In recent years, many novel techniques for SLN mapping have been developed including fluorescence, x-ray, and magnetic resonant detection. Whilst each technique has its own advantages/disadvantages, the role of targeted contrast agents (for enhanced retention in the SLN, or for immunostaging) is increasing, and may represent the new standard for mapping the SLN in many solid organ tumours. This review article discusses current limitations of conventional techniques, limiting factors of nanoparticulate based contrast agents, and efforts to circumvent these limitations with modern tracer architecture.     

Three stage axillary lymphatic massage optimizes sentinel lymph node localisation using blue dye

Aims

This paper describes a simple technique of axillary and breast massage which improves the successful identification of blue sentinel nodes using patent blue dye alone.

Methods

Patent blue dye was injected in the subdermal part of the retroaroelar area in 167 patients having surgical treatment for invasive breast cancer. Three stage axillary lymphatic massage was performed prior to making the axillary incision for sentinel lymph node biopsy. All patients had completion axillary sampling or clearance.

Results

A blue lymphatic duct leading to lymph nodes of the first drainage was identified in 163 (97%) of the patients. Results are compared with 168 patients who had sentinel lymph node biopsy using blue dye without axillary massage. Allergic reactions were observed in four patients (1.2%).

Conclusion

Three stage axillary lymphatic massage improves the successful identification of a blue sentinel lymph node in breast cancer patients.

     

Patterns of DNA copy number changes in sentinel lymph node breast cancer metastases

The sentinel lymph node (SLN) is considered to be the first axillary node that contains malignant cells in metastatic breast tumors, and its positivity is currently used in clinical practice as an indication for axillary lymph node dissection. Therefore, accurate evaluation of the SLN for the presence of breast metastatic cells is essential. The main aim of our study is to characterize the genomic changes present in the SLN metastatic samples with the ultimate goal of improving the predictive value of SLN evaluation. Twenty paired samples of SLN metastases and their corresponding primary breast tumors (PBT) were investigated for DNA copy number changes using comparative genomic hybridization (CGH). Non-random DNA copy number changes were observed in all the lesions analyzed, with gains being more common than losses. In 75% of the cases there was at least one change common to both PBT and SLN. The most frequent changes detected in both lesions were gains of 1pter-->p32, 16, 17, 19, and 20 and losses of 6q13-->q23 and 13q13-->q32. In the PBT group, alterations on chromosomes 1, 16, and 20 were the most frequent, whereas chromosomes 1, 6, and 19 were the ones with the highest number of changes in the SLN metastatic group. A positive correlation was found between the DNA copy number changes per chromosome in each of the groups. Our findings indicate the presence of significant DNA copy number changes in the SLN metastatic lesions that could be used in the future as additional markers to improve the predictive value of SLN biopsy procedure.     

Clinical evaluation of a novel intraoperative handheld gamma camera for sentinel lymph node biopsy

ObjectivePreoperative lymphoscintigraphy (PLS) combined with intraoperative gamma probe (GP) localization is standard procedure for localizing the sentinel lymph nodes (SLN) in melanoma and breast cancer. In this study, we evaluated the ability of a novel intraoperative handheld gamma camera (IHGC) to image SLNs during surgery.MethodsThe IHGC is a small-field-of-view camera optimized for real-time imaging of lymphatic drainage patterns. Unlike conventional cameras, the IHGC can acquire useful images in a few seconds in a free-running fashion and be moved manually around the patient to find a suitable view of the node. Thirty-nine melanoma and eleven breast cancer patients underwent a modified SLN biopsy protocol in which nodes localized with the GP were imaged with the IHGC. The IHGC was also used to localize additional nodes that could not be found with the GP.ResultsThe removal of 104 radioactive SLNs was confirmed ex vivo by GP counting. In vivo, the relative node detection sensitivity was 88.5 (82.3, 94.6)% for the IHGC (used in conjunction with the GP) and 94.2 (89.7, 98.7)% for the GP alone, a difference not found to be statistically significant (McNemar test, p = 0.24).ConclusionSmall radioactive SLNs can be visualized intraoperatively using the IHGC with exposure time of 20 s or less, with no significant difference in node detection sensitivity compared to a GP. The IHGC is a useful complement to the GP, especially for SLNs that are difficult to locate with the GP alone.     

Adenovirus-mediated gene expression imaging to directly detect sentinel lymph node metastasis of prostate cancer

The accurate assessment of nodal involvement in prostate cancer is crucial to planning treatment, yet there is a shortage of noninvasive imaging techniques capable of visualizing nodal lesions directly. This study demonstrates the feasibility of using recombinant human adenoviral vectors to detect nodal metastases in a human prostate cancer model. This was achieved by the prostate-restricted expression of optical and positron emission tomography (PET) imaging reporter genes by the viral vector coupled with the innate lymphotropic properties of adenovirus. We show that peritumoral administration of these vectors results in the direct detection of reporter gene expression in metastatic lesions within sentinel lymph nodes. Notably, this approach parallels the current lymphoscintigraphy method but enables the direct PET visualization of sentinel lymph node metastases, eliminating the need for invasive lymphadenectomy. These findings may lead to more effective diagnostic and therapeutic strategies for individuals with advanced-stage prostate cancer.     

The effect of mannosylation of liposome-encapsulated indocyanine green on imaging of sentinel lymph node

Abstract

The imaging of sentinel lymph nodes (SLN) has been researched for its role in assessing cancer progression and postsurgical lymphedema. Indocyanine green (ICG) is a near-infrared (NIR) optical dye that has been approved by the Food and Drug Administration. It is known that liposome-encapsulated ICG (LP-ICG) has improved stability and fluorescence signal compared with ICG. We designed mannosylated liposome-encapsulated ICG (M-LP-ICG) as an optical contrast agent for SLN. M-LP-ICG has a higher UV absorbance spectrum and fluorescence intensity than LP-ICG. The stability of M-LP-ICG measured in 50% fetal bovine serum solution by a dialysis method was better than that of LP-ICG. M-LP-ICG demonstrated a high uptake in RAW 264.7 macrophage cell because the density of mannose is high. There were differences between M-LP-ICG and glucosylated liposome-encapsulated ICG (G-LP-ICG), which are geometrical isomers. The result of an inhibition study of M-LP-ICG showed a statistically significant decrease in uptake in RAW 264.7 cells after either co-treatment or pre-treatment with d-(+)-mannose as an inhibitor. Results from an in vitro experiment demonstrated that M-LP-ICG was specifically taken up by macrophage cells through the mannose receptor on its surface. The time-series images acquired from a normal mouse model after subcutaneous injection showed that the signal from M-LP-ICG in SLN and other organs appeared early and disappeared quickly in comparison with signals from LP-ICG. Not only the sentinel but also the draining lymph nodes were observed partly in M-LP-ICG. M-LP-ICG appears to increase the specificity of uptake and retention in macrophages, making it a good candidate contrast agent for an optic imaging system for SLN and the lymphatic system.