Local delivery of low-dose anti-CTLA-4 to the melanoma lymphatic basin leads to systemic Treg reduction and effector T cell activation.

Preclinical studies show that locoregional CTLA-4 blockade is equally effective in inducing tumor eradication as systemic delivery, without the added risk of immune-related side effects. This efficacy is related to access of the CTLA-4 blocking antibodies to tumor-draining lymph nodes (TDLNs). Local delivery of anti-CTLA-4 after surgical removal of primary melanoma, before sentinel lymph node biopsy (SLNB), provides a unique setting to clinically assess the role of TDLN in the biological efficacy of locoregional CTLA-4 blockade. Here, we have evaluated the safety, tolerability, and immunomodulatory effects in the SLN and peripheral blood of a single dose of tremelimumab [a fully human immunoglobulin gamma-2 (IgG2) mAb directed against CTLA-4] in a dose range of 2 to 20 mg, injected intradermally at the tumor excision site 1 week before SLNB in 13 patients with early-stage melanoma (phase 1 trial; NCT04274816). Intradermal delivery was safe and well tolerated and induced activation of migratory dendritic cell (DC) subsets in the SLN. It also induced profound and durable decreases in regulatory T cell (Treg) frequencies and activation of effector T cells in both SLN and peripheral blood. Moreover, systemic T cell responses against NY-ESO-1 or MART-1 were primed or boosted (N = 7), in association with T cell activation and central memory T cell differentiation. These findings indicate that local administration of anti-CTLA-4 may offer a safe and promising adjuvant treatment strategy for patients with early-stage melanoma. Moreover, our data demonstrate a central role for TDLN in the biological efficacy of CTLA-4 blockade and support TDLN-targeted delivery methods.

Cumulative 5-year Results of a Randomized Controlled Trial Comparing Biological Mesh With Primary Perineal Wound Closure After Extralevator Abdominoperineal Resection (BIOPEX-study).

OBJECTIVE: To determine long-term outcomes of a randomized trial (BIOPEX) comparing biological mesh and primary perineal closure in rectal cancer patients after extralevator abdominoperineal resection and preoperative radiotherapy, with a primary focus on symptomatic perineal hernia. SUMMARY BACKGROUND DATA: BIOPEX is the only randomized trial in this field, which was negative on its primary endpoint (30-day wound healing). METHODS: This was a posthoc secondary analysis of patients randomized in the BIOPEX trial to either biological mesh closure (n = 50; 2 dropouts) or primary perineal closure (n = 54; 1 dropout). Patients were followed for 5 years. Actuarial 5-year probabilities were determined by the Kaplan-Meier statistic. RESULTS: Actuarial 5-year symptomatic perineal hernia rates were 7% (95% CI, 0-30) after biological mesh closure versus 30% (95% CI, 10-49) after primary closure (P = 0.006). One patient (2%) in the biomesh group underwent elective perineal hernia repair, compared to 7 patients (13%) in the primary closure group (P = 0.062). Reoperations for small bowel obstruction were necessary in 1/48 patients (2%) and 5/53 patients (9%), respectively (P = 0.208). No significant differences were found for chronic perineal wound problems, locoregional recurrence, overall survival, and main domains of quality of life and functional outcome. CONCLUSIONS: Symptomatic perineal hernia rate at 5-year follow-up after abdominoperineal resection for rectal cancer was significantly lower after biological mesh closure. Biological mesh closure did not improve quality of life or functional outcomes.

Breast cancer-induced immune suppression in the sentinel lymph node is effectively countered by CpG-B in conjunction with inhibition of the JAK2/STAT3 pathway.

BACKGROUND: We previously showed selectively hampered activation of lymph node-resident (LNR) dendritic cell (DC) subsets in the breast cancer (BrC) sentinel lymph node (SLN) to precede a state of profound T cell anergy. Reactivating these DC subsets by intratumoral delivery of the Toll-like receptor-9 (TLR9) agonist CpG-B could potentially offer a promising immune therapeutic strategy to combat this immune suppression and prevent disease spread. Unfortunately, CpG-B can limit its own immune stimulatory activity through direct TLR9-mediated activation of signal transducer and activator of transcription 3 (STAT3), pinpointed as a key regulator of immune suppression in the tumor microenvironment. Here, we have investigated whether in vitro exposure to CpG-B, with or without simultaneous inhibition of STAT3 signaling, could overcome immune suppression in BrC SLN. METHODS: Immune modulatory effects of CpG-B (CPG7909) with or without the JAK2/STAT3 inhibitor (STAT3i) AG490 were assessed in ex vivo cultured BrC SLN-derived single-cell suspensions (N=29). Multiparameter flow cytometric analyses were conducted for DC and T cell subset characterization and assessment of (intracellular) cytokine profiles. T cell reactivity against the BrC-associated antigen Mammaglobin-A was determined by means of interferon-γ ELISPOT assay. RESULTS: Although CpG-B alone induced activation of all DC subsets, combined inhibition of the JAK2/STAT3 pathway resulted in superior DC maturation (ie, increased CD83 expression), with most profound activation and maturation of LNR DC subsets. Furthermore, combined CpG-B and JAK2/STAT3 inhibition promoted Th1 skewing by counterbalancing the CpG-induced Th2/regulatory T cell response and significantly enhanced Mammaglobin-A specific T cell reactivity. CONCLUSION: Ex vivo immune modulation of the SLN by CpG-B and simultaneous JAK2/STAT3 inhibition can effectively overcome BrC-induced immune suppression by preferential activation of LNR DC, ultimately restoring type 1-mediated antitumor immunity, thereby securing a BrC-specific T cell response. These findings provide a clear rationale for clinical exploration of SLN-immune potentiation through local CpG/STAT3i administration in patients with BrC.

Perineal wound closure using gluteal turnover flap or primary closure after abdominoperineal resection for rectal cancer: study protocol of a randomised controlled multicentre trial (BIOPEX-2 study).

BACKGROUND: Abdominoperineal resection (APR) for rectal cancer is associated with high morbidity of the perineal wound, and controversy exists about the optimal closure technique. Primary perineal wound closure is still the standard of care in the Netherlands. Biological mesh closure did not improve wound healing in our previous randomised controlled trial (BIOPEX-study). It is suggested, based on meta-analysis of cohort studies, that filling of the perineal defect with well-vascularised tissue improves perineal wound healing. A gluteal turnover flap seems to be a promising method for this purpose, and with the advantage of not having a donor site scar. The aim of this study is to investigate whether a gluteal turnover flap improves the uncomplicated perineal wound healing after APR for rectal cancer. METHODS: Patients with primary or recurrent rectal cancer who are planned for APR will be considered eligible in this multicentre randomised controlled trial. Exclusion criteria are total exenteration, sacral resection above S4/S5, intersphincteric APR, biological mesh closure of the pelvic floor, collagen disorders, and severe systemic diseases. A total of 160 patients will be randomised between gluteal turnover flap (experimental arm) and primary closure (control arm). The total follow-up duration is 12 months, and outcome assessors and patients will be blinded for type of perineal wound closure. The primary outcome is the percentage of uncomplicated perineal wound healing on day 30, defined as a Southampton wound score of less than two. Secondary outcomes include time to perineal wound closure, incidence of perineal hernia, the number, duration and nature of the complications, re-interventions, quality of life and urogenital function. DISCUSSION: The uncomplicated perineal wound healing rate is expected to increase from 65 to 85% by using the gluteal turnover flap. With proven effectiveness, a quick implementation of this relatively simple surgical technique is expected to take place. TRIAL REGISTRATION: The trial was retrospectively registered at Clinicaltrials.gov NCT04004650 on July 2, 2019.

In the mix: the potential benefits of adding GM-CSF to CpG-B in the local treatment of patients with early-stage melanoma.

Whereas TLR9 agonists are recognized as powerful stimulators of antitumor immunity, GM-CSF has had mixed reviews. In previously reported randomized trials we assessed the effects of local immune modulation in early-stage melanoma with CpG-B alone or with GM-CSF. Here we discuss the added value of GM-CSF and show sex-related differences.

Selectively hampered activation of lymph node-resident dendritic cells precedes profound T cell suppression and metastatic spread in the breast cancer sentinel lymph node.

BACKGROUND: Immune regulated pathways influence both breast cancer (BrC) development and response to (neo)adjuvant chemotherapy. The sentinel lymph node (SLN), as the first metastatic site, is also the first site where BrC-induced suppression of immune effector subsets occurs. Since intricate knowledge of the phenotypic and functional status of these immune effector subsets is lacking, we set out to map the immune landscape of BrC SLN. METHODS: Viable LN cells from BrC SLN (n = 58) were used for detailed flowcytometry-assisted mapping of the immune landscape of BrC SLN in a comparative analysis with healthy (i.e. prophylactic mastectomy-derived) axillary lymph nodes (HLN, n = 17). Findings were related to clinicopathological characteristics. RESULTS: Our data show that BrC-induced immune suppression in tumor-involved SLN, as evidenced by increased Treg and MDSC rates as well as by a generalized state of T cell anergy, coincides with hampered activation of LN-resident (LNR) dendritic cell (DC) subsets rather than of migratory DC subsets. Importantly, suppression of these LN-resident DC subsets preceded profoundly disabled T cell effector functions in tumor-involved SLN. Furthermore, we provide evidence that the suppressed state of LNR-cDC is not only related to nodal involvement but is also related to high-risk breast cancer subtypes that lack expression of hormone receptors and may be a negative predictor of disease-free survival. CONCLUSION: These data thus provide new insights in the mechanisms underlying loco-regional immune suppression induced by BrC and how these relate to clinical outcome. They identify the LNR-cDC subset as a pivotal regulatory node in cellular immune suppressive pathways and therefore as a promising therapeutic target to combat immune suppression and secure the induction of effective antitumor immunity, e.g. in combination with neo-adjuvant chemotherapy. .

Local Adjuvant Treatment with Low-Dose CpG-B Offers Durable Protection against Disease Recurrence in Clinical Stage I-II Melanoma: Data from Two Randomized Phase II Trials.

Purpose: Although risk of recurrence after surgical removal of clinical stage I-II melanoma is considerable, there is no adjuvant therapy with proven efficacy. Here, we provide clinical evidence that a local conditioning regimen, aimed at immunologic arming of the tumor-draining lymph nodes, may provide durable protection against disease recurrence (median follow-up, 88.8 months).Experimental Design: In two randomized phase II trials, patients, diagnosed with stage I-II melanoma after excision of the primary tumor, received local injections at the primary tumor excision site within 7 days preceding re-excision and sentinel lymph node (SLN) biopsy of either a saline placebo (n = 22) or low-dose CpG type B (CpG-B) with (n = 9) or without (n = 21) low-dose GM-CSF.Results: CpG-B treatment was shown to be safe, to boost locoregional and systemic immunity, to be associated with lower rates of tumor-involved SLN (10% vs. 36% in controls, P = 0.04), and, at a median follow-up of 88.8 months, to profoundly improve recurrence-free survival (P = 0.008), even for patients with histologically confirmed (i.e., pathologic) stage I-II disease (P = 0.02).Conclusions: Potentially offering durable protection, local low-dose CpG-B administration in early-stage melanoma provides an adjuvant treatment option for a large group of patients currently going untreated despite being at considerable risk for disease recurrence. Once validated in a larger randomized phase III trial, this nontoxic immunopotentiating regimen may prove clinically transformative. Clin Cancer Res; 23(19); 5679-86. ©2017 AACR.

Biological Mesh Closure of the Pelvic Floor After Extralevator Abdominoperineal Resection for Rectal Cancer: A Multicenter Randomized Controlled Trial (the BIOPEX-study).

OBJECTIVE: To determine the effect of biological mesh closure on perineal wound healing after extralevator abdominoperineal resection (eAPR). BACKGROUND: Perineal wound complications frequently occur after eAPR with preoperative radiotherapy for rectal cancer. Cohort studies have suggested that biological mesh closure of the pelvic floor improves perineal wound healing. METHODS: Patients were randomly assigned to primary closure (standard arm) or biological mesh closure (intervention arm). A non-cross-linked porcine acellular dermal mesh was sutured to the pelvic floor remnants in the intervention arm, followed by a layered closure of the ischioanal and subcutaneous fat and skin similar to the control intervention. The outcome of the randomization was concealed from the patient and perineal wound assessor. The primary endpoint was the rate of uncomplicated perineal wound healing defined as a Southampton wound score of less than 2 at 30 days postoperatively. Patients were followed for 1 year. RESULTS: In total, 104 patients were randomly assigned to primary closure (n = 54; 1 dropouts) and biological mesh closure (n = 50; 2 dropouts). Uncomplicated perineal wound healing rate at 30 days was 66% (33/50; 3 not evaluable) after primary closure, which did not significantly differ from 63% (30/48) after biological mesh closure [relative risk 1.056; 95% confidence interval (CI) 0.7854-1.4197; P = 0.7177). Freedom from perineal hernia at 1 year was 73% (95% CI 60.93-85.07) versus 87% (95% CI 77.49-96.51), respectively (P = 0.0316). CONCLUSIONS: Perineal wound healing after eAPR with preoperative radiotherapy for rectal cancer was not improved when using a biological mesh. A significantly lower 1-year perineal hernia rate after biological mesh closure is a promising secondary finding that needs longer follow-up to determine its clinical relevance.

Randomized controlled multicentre study comparing biological mesh closure of the pelvic floor with primary perineal wound closure after extralevator abdominoperineal resection for rectal cancer (BIOPEX-study).

BACKGROUND: Primary perineal wound closure after conventional abdominoperineal resection (cAPR) for rectal cancer has been the standard of care for many years. Since the introduction of neo-adjuvant radiotherapy and the extralevator APR (eAPR), oncological outcome has been improved, but at the cost of increased rates of perineal wound healing problems and perineal hernia. This has progressively increased the use of biological meshes, although not supported by sufficient evidence. The aim of this study is to determine the effectiveness of pelvic floor reconstruction using a biological mesh after standardized eAPR with neo-adjuvant (chemo)radiotherapy compared to primary perineal wound closure. METHODS/DESIGN: In this multicentre randomized controlled trial, patients with a clinical diagnosis of primary rectal cancer who are scheduled for eAPR after neo-adjuvant (chemo)radiotherapy will be considered eligible. Exclusion criteria are prior radiotherapy, sacral resection above S4/S5, allergy to pig products or polysorbate, collagen disorders, and severe systemic diseases affecting wound healing, except for diabetes. After informed consent, 104 patients will be randomized between standard care using primary wound closure of the perineum and the experimental arm consisting of suturing a biological mesh derived from porcine dermis in the pelvic floor defect, followed by perineal closure similar to the control arm. Patients will be followed for one year after the intervention and outcome assessors and patients will be blinded for the study treatment. The primary endpoint is the percentage of uncomplicated perineal wound healing, defined as a Southampton wound score of less than II on day 30. Secondary endpoints are hospital stay, incidence of perineal hernia, quality of life, and costs. DISCUSSION: The BIOPEX-study is the first randomized controlled multicentre study to determine the additive value of using a biological mesh for perineal wound closure after eAPR with neo-adjuvant radiotherapy compared to primary perineal wound closure with regard to perineal wound healing and the occurrence of perineal hernia. TRAIL REGISTRATION NUMBER: NCT01927497 (Clinicaltrial.gov).

Feasibility of flowcytometric quantitation of immune effector cell subsets in the sentinel lymph node of the breast after cryopreservation.

The sentinel lymph node (SLN) is an emerging focus for immunological research in breast cancer. Cryopreservation of SLN single-cell suspensions allows for simultaneous phenotypic multi-parameter analyses and minimizes operator dependent variability. This is of particular importance for immunomonitoring of large multicenter trials. However, little data are available regarding the influence of cryopreservation on phenotypic characteristics of lymph node dendritic cells and T cells. In this study we assessed the feasibility of cryopreservation of viable SLN cell samples for flowcytometric analysis, by comparing quantitative analyses of SLN cell samples after freeze-thawing with direct analysis of fresh SLN cell samples. SLN were collected from nine breast cancer patients. From each SLN cell sample, half was used for immediate analysis and half was analyzed after cryopreservation and thawing. Conventional dendritic cell (cDC) and T cell subsets were quantified and phenotypically characterized by flow cytometry. The observed frequencies of both CD1a(+) and CD1a(-)CD11c(+)CD14(-) cDC subsets showed significant correlation between the fresh and frozen-thawed samples. Similar high correlations were found for CD83 and CD86 expression markers on the more frequent (>0.2%) CD1a(+) and CD1a(-)CD11c(+)CD14(-) cDC subset, but not on the low-frequency (<0.2%) CD1a(+)CD11c(+)CD14(+) cDC subset. CD4/CD8 T cell ratios were comparable and were significantly correlated pre- and post-freezing. Regulatory CD4(+)CD25(hi) T cell frequencies and their FoxP3 expression levels were significantly higher after freezing-thawing than in the freshly analyzed samples. Nevertheless, a highly significant correlation was found for both parameters pre- and post-freezing. Cryopreservation and thawing seems a valid and practical alternative to direct analysis of fresh viable lymph node cells, without introducing cryo-dependent variance between SLN samples. However, enumeration of low-frequency cell populations and assessment of their marker expression levels are less reliable after cryopreservation and should be assessed and considered in the design of each clinical trial.

Preoperative granulocyte/macrophage colony-stimulating factor (GM-CSF) increases hepatic dendritic cell numbers and clustering with lymphocytes in colorectal cancer patients.

Despite surgery with curative intent, approximately 30% of colorectal carcinoma patients will develop liver metastases during follow-up. Synchronous occult micrometastases, tumor cell shedding into the portal circulation and postoperative immune impairment have all been suggested to facilitate outgrowth of liver metastases. In experimental models, increases in both number of resident macrophages of the liver, the so-called Kupffer cells (KC), and tumoricidal capacity of KC were observed after pretreatment with granulocyte/macrophage colony-stimulating factor (GM-CSF), a potent immuno-stimulatory agent. Following perioperative recombinant human GM-CSF (rhGM-CSF), we previously showed activation of the systemic immune response in the postoperative period, which is normally transiently down-modulated after surgery. Therefore, in this pilot study, effects of preoperative rhGM-CSF administration on the composition of human liver immune cell population were evaluated in patients undergoing surgery for colorectal cancer. No difference in KC numbers of rhGM-CSF-treated patients was observed. Importantly, however, a 6-fold increase in dendritic cell (DC) numbers was observed compared to control patients, as quantified by immunohistochemistry of liver biopsies, taken during laparotomy. Furthermore, direct contact between liver CD8+ cells and DC was significantly enhanced in rhGM-CSF-treated patients. Both increases in DC numbers and DC interaction with CD8+ T cells suggest enhanced immunological activation, which may reduce liver metastases formation and ultimately improve survival after initial colorectal surgery.

Tumor-specific CD8+ T cell reactivity in the sentinel lymph node of GM-CSF-treated stage I melanoma patients is associated with high myeloid dendritic cell content.

PURPOSE: Impaired immune functions in the sentinel lymph node (SLN) may facilitate early metastatic events during melanoma development. Local potentiation of tumor-specific T cell reactivity may be a valuable adjuvant treatment option. EXPERIMENTAL DESIGN: We examined the effect of locally administered granulocyte/macrophage-colony stimulating factor (GM-CSF) on the frequency of tumor-specific CD8+ T cells in the SLN and blood of patients with stage I melanoma. Twelve patients were randomly assigned to preoperative local administration of either recombinant human GM-CSF or NaCl 0.9%. CD8+ T cells from SLN and peripheral blood were tested for reactivity in an IFNgamma ELISPOT assay against the full-length MART-1 antigen and a number of HLA-A1, HLA-A2, and HLA-A3-restricted epitopes derived from a range of melanoma-associated antigens. RESULTS: Melanoma-specific CD8+ T cell response rates in the SLN were one of six for the control group and four of six for the GM-CSF-administered group. Only one patient had detectable tumor-specific CD8+ T cells in the blood, but at lower frequencies than in the SLN. All patients with detectable tumor-specific CD8+ T cells had a percentage of CD1a+ SLN-dendritic cells (DC) above the median (i.e., 0.33%). This association between above median CD1a+ SLN-DC frequencies and tumor antigen-specific CD8+ T cell reactivity was significant in a two-sided Fisher's exact test (P = 0.015). CONCLUSIONS: Locally primed antitumor T cell responses in the SLN are detectable as early as stage I of melanoma development and may be enhanced by GM-CSF-induced increases in SLN-DC frequencies.

Matched skin and sentinel lymph node samples of melanoma patients reveal exclusive migration of mature dendritic cells.

Mature and immature myeloid dendritic cells (DCs) are thought to differentially modulate T-cell responses in secondary lymphoid tissues. Although mature DCs are believed to induce T-cell activation under proinflammatory conditions, immature DCs are believed to maintain a state of T-cell tolerance under steady state conditions. However, little is known about the actual activation state of human DCs under these different conditions. Here, we compare the frequency and activation state of human DCs between matched skin and sentinel lymph node (SLN) samples, after intradermal administration of either granulocyte/macrophage colony-stimulating factor (GM-CSF) or saline, at the excision site of stage I primary melanoma. Although DCs remained immature (CD1a+CD83-) and mostly situated in the epidermis of the saline-injected skin (fully consistent with a quiescent steady state), mature (CD1a+CD83+) DC frequencies significantly increased in the GM-CSF-injected skin and correlated with the number of mature DCs in the SLN, indicative of increased DC migration. Interestingly, irrespective of GM-CSF or saline administration, all CD1a+ myeloid DCs in the SLN were phenotypically mature (ie, CD83+). These data are indicative of migration of small numbers of phenotypically mature DCs to lymph nodes under steady state conditions.

Sentinel lymph node tumor load: an independent predictor of additional lymph node involvement and survival in melanoma.

BACKGROUND: Even though 60% to 80% of melanoma patients with a positive sentinel lymph node (SLN) have no positive additional lymph nodes (ALNs), all these patients are subjected to an ALN dissection (ALND) with its associated morbidity. The aim of this study was to predict the absence of ALN metastases in patients with a positive SLN by using features of the primary melanoma and SLN tumor load. METHODS: Of 71 SLN-positive patients, 52 had metastasis limited to the SLN (group 1), and 19 had > or =1 positive ALN after ALND (group 2). The tumor load of the SLN was assessed by measuring the total surface area by computerized morphometry. Breslow thickness, ulceration and lymphatic invasion of the primary tumor, and total SLN metastatic area were tested as covariates predicting the absence of positive ALNs. RESULTS: The mean SLN metastatic area was 1.18 mm(2) (group 1) and 3.39 mm(2) (group 2) (P = .003) and was the only significant and independent factor after multivariate analysis (P = .02). None of the patients with both a Breslow thickness <2.5 mm and an SLN metastatic area <.3 mm(2) had a positive ALN. CONCLUSIONS: SLN metastatic area can be used to predict the absence of positive ALNs in melanoma patients. In this study, patients with a Breslow thickness <2.5 mm and an SLN tumor load <.3 mm(2 )seemed to have no positive ALN and had excellent survival. We hypothesize that this subgroup might not benefit from ALND. Prospective larger trials, using this model and randomizing between ALND and no ALND, should confirm this hypothesis.

Local administration of granulocyte/macrophage colony-stimulating factor increases the number and activation state of dendritic cells in the sentinel lymph node of early-stage melanoma.

The initial tumor-draining lymph node, the sentinel lymph node, not only constitutes the first expected site of micrometastasis but also the first point of contact between tumor-associated antigens and the adaptive immune system. A tumor-induced decrease in the frequency and activation state of sentinel lymph node dendritic cells will impair the generation of effective antitumor T-cell responses and increase the likelihood of metastatic spread. Here, we demonstrate that intradermal administration of granulocyte macrophage-colony stimulating factor around the excision site of stage I primary melanoma tumors increases the number and activation state of dendritic cells in the paracortical areas of the sentinel lymph node and enhances their binding to T cells. We conclude that local treatment of melanoma patients with granulocyte macrophage-colony stimulating factor, before surgery, conditions the sentinel lymph node microenvironment to enhance mature dendritic cell recruitment and hypothesize that this may be more conducive to the generation of T-cell-mediated antitumor immunity.

Pattern and incidence of first site recurrences following sentinel node procedure in melanoma patients.

Studies of large series of melanoma patients indicated that the average incidence of developing a recurrence during follow-up was 40%. The most frequent first sites of these recurrences were the regional lymph nodes. We hypothesized that the sentinel node (SN) procedure may change the pattern of recurrence by reducing the number of first recurrences in the regional lymph node basin during follow-up to a negligible number, and that locoregional cutaneous and distance metastases are the major future sites of recurrence. We further studied the influence of SN status together with different influential factors on prognosis. An SN procedure with a triple technique was performed in 250 consecutive patients with proven AJCC stages I and II cutaneous melanoma. The median follow-up was 38 months. So far, 44 patients (18%) have developed a recurrence of the disease. The distribution of localization of the first metastases was as follows: 23 patients (52%) with a locoregional cutaneous recurrence; 4 (9%) with recurrence in the regional lymph node basin; 2 (5%) with recurrence in an interval node; and 15 (34%) with distant recurrence. The relative risk of developing recurrence for SN-positive patients is 4.2; for Breslow thickness of 1.51 to 4.00 mm it is 5.5, and thicker than 4.0 mm it is 6.2; for lymphatic invasion 7.6; and for ulceration 3.8. We conclude that the SN procedure changes the pattern of recurrences during follow-up by reducing the number of first recurrences within the regional lymph node basin to a negligible number. High Breslow thickness, lymphatic invasion, and ulceration of the primary melanoma are strong risk factors for recurrence.

Sampling tumor-draining lymph nodes for phenotypic and functional analysis of dendritic cells and T cells.

Immune responses against tumor antigens will initially occur in the first tumor-draining lymph node, the sentinel node (SN). Because of extensive diagnostic procedures, obtaining a piece of SN to isolate viable immune cells for functional analyses is often impossible. For this reason an alternative method to obtain viable cells from a lymph node (LN) was investigated, ie, scraping LNs with a surgical blade, and compared with dissociation of total LNs. Tumor-draining lymph nodes were retrieved from five oncological patients. The collected dendritic cells and T cells were phenotypically and functionally characterized by flow cytometry and antigen-specific interferon (IFN)-gamma release in an ELISPOT assay. Results were compared between the two isolation methods. Viabilities and phenotypic characteristics of the collected cells were entirely comparable for both methods. T-cell functionality was also comparable between both methods, with equal T-cell expansion factors and similar frequencies of cytotoxic T cells specifically recognizing the M1 matrix protein of Influenza haemophilus or the tumor antigen Her-2/neu. In conclusion, scraping LNs to obtain cells for analysis of immune functions in LNs is feasible and presents a good alternative to dissociation of LNs. Scraping may even be applied to small LNs that a pathologist will submit entirely for histological examination and may thus prove useful in the monitoring of immune responses in SNs.