Extramammary Paget disease (EMPD) is an uncommon adenocarcinoma of apocrine gland-rich areas, presenting significant diagnostic challenges due to its nonspecific clinical appearance and frequent misidentification as benign, inflammatory skin conditions. Traditional diagnostic methods such as biopsy are invasive and uncomfortable, often required repeatedly due to high recurrence rates. Dermoscopy and non-invasive imaging techniques have been used but provide limited diagnostic accuracy due to their constraints in depth penetration and resolution. Recent advancements in imaging technologies, such as line-field confocal optical coherence tomography (LC-OCT), show promise in enhancing diagnostic precision while minimizing invasive procedures. LC-OCT merges high-resolution imaging with deep penetration capabilities, capturing detailed horizontal and vertical skin images akin to histopathology. This study evaluated the diagnostic performance of LC-OCT in detecting EMPD and its recurrence in 17 clinically suspicious anogenital regions, belonging to six patients. Data were collected prospectively at the patient’s bedside by an LC-OCT expert with poor training for EMPD, and, then, reviewed retrospectively by an independent LC-OCT expert with adequate training for EMPD and no concerns about time. The prospective examination yielded 64.7% accuracy (11 true results out of 17 total cases), 71.4% sensitivity (10 true positives out of 14 actual positives), and 33.3% specificity (1 true negative out of 3 actual negatives). The retrospective analysis achieved 94.1% accuracy (16 true results out of 17 total cases), 100% sensitivity (14 true positives out of 14 actual positives), and 66.7% specificity (2 true positives out of 3 actual positives), with the only false positive case being a difficult-to-diagnose concomitant presentation of a lichen sclerosus et atrophicus. Despite the need for specialized training, our results suggest that LC-OCT represents a valuable tool for accurately identifying EMPD and improving its management by reducing unnecessary biopsies. Further studies are needed to standardize its clinical application.

BACKGROUND:Treatment with anti-PD1 monoclonal antibodies improves the survival of metastatic melanoma patients but only a subgroup of patients benefits from durable disease control. Predictive biomarkers for durable benefit could improve the clinical management of patients. METHODS:Plasma samples were collected from patients receiving anti-PD1 therapy for ctDNA quantitative assessment of BRAFV600 and NRASQ61/G12/G13 mutations. RESULTS:After a median follow-up of 84 weeks 457 samples from 85 patients were analyzed. Patients with undetectable ctDNA at baseline had a better PFS (Hazard ratio (HR) = 0.47, median 26 weeks versus 9 weeks, p = 0.01) and OS (HR = 0.37, median not reached versus 21.3 weeks, p = 0.005) than patients with detectable ctDNA. Additionally, the HR for death was lower after the ctDNA level became undetectable during follow-up (adjusted HR: 0.16 (95% CI 0.07-0.36), p-value < 0.001). ctDNA levels > 500 copies/ml at baseline or week 3 were associated with poor clinical outcome. Patients progressive exclusively in the central nervous system (CNS) had undetectable ctDNA at baseline and at subsequent assessments. In multivariate analysis adjusted for LDH, CRP, ECOG and number of metastatic sites, the ctDNA remained significant for PFS and OS. A positive correlation was observed between ctDNA levels and total metabolic tumor volume (TMTV), number of metastatic sites and total tumor burden. CONCLUSIONS:Assessment of ctDNA baseline and during therapy was predictive for tumor response and clinical outcome in metastatic melanoma patients and reflected the tumor burden. ctDNA evaluation provided reliable complementary information during anti-PD1 antibody therapy.

Monoclonal antibodies that block the programmed death-1 (anti-PD-1) or cytotoxic T-lymphocyte antigen-4 (CTLA-4) immune checkpoint receptors (pembrolizumab, nivolumab, ipilimumab, or the combination of nivolumab with ipilimumab) are approved treatment option for patients with advanced melanoma. Over half of all patients are refractory to these immunotherapies and are in need of alternative or complementary treatment options. Talimogene laherparepvec (T-VEC) is a first-in-class intralesionally delivered oncolytic immunotherapy, which has proven efficacy in the treatment of advanced melanoma. A proportion of patients treated with T-VEC will benefit from an abscopal response of noninjected metastases indicative of a systemic antitumor immune response elicited by the intratumoral injections. At present it remains unknown whether the systemic antitumor responses elicited by T-VEC are nonredundant with immune-checkpoint blockade. Recent data on potential synergy between T-VEC and both PD-1 and CTLA-4 blockade suggest that the mechanism of action may be complementary. We report on the successful treatment with intralesional T-VEC of two female patients with locoregionally advanced BRAF V600 wild-type melanoma who previously progressed on anti-PD-1 and anti-CTLA-4 inhibitors.

s presented at the 24th Annual Congress of the Belgian Society of Internal Medicine, 13–14 December 2019, Dolce La Hulpe, La Hulpe, Belgium Oral Communication – Case Reports 24 IgA nephropathy associated with thrombotic microangiopathy and diffuse alveolar hemorrhage. Quentin Binet, Christian Miesse, Benoît Colinet, Selda Aydin, Jean-Philippe Lengelé, JeanFrançois Cambier, Grand Hôpital de Charleroi, Gilly, Belgium, Cliniques Universitaires Saint-Luc,

Anti-programmed death 1 (PD-1) monoclonal antibodies improve the survival of metastatic melanoma patients. Predictive or monitoring biomarkers for response to this therapy could improve the clinical management of these patients. To date, no established biomarkers are available for monitoring the response to immunotherapy. Tumor- specific mutations in circulating tumor DNA (ctDNA) such as BRAF and NRAS mutations for melanoma patients have been proposed for monitoring of immunotherapy response. We present seven illustrative cases for the use of ctDNA BRAF and NRAS mutations' monitoring in plasma. The cases described exemplify four distinct clinical benefit patterns: rapid and durable complete response (CR), early progression, followed by CR, CR followed by early progression after interrupting treatment and long-term disease stabilization. These representative cases suggest that comprehensive BRAF/NRAS ctDNA monitoring during anti-PD1 therapy is informative and can be of added value for the monitoring of melanoma patients gaining clinical benefit on anti-PD1 treatment. An important advantage of our approach is that using the cartridge system on the Idylla platform for mutation analysis, the results become available the same day 2 h after plasma collection. Therefore, in the future, the ctDNA level can be an element in the clinical management of the patients.

Ipilimumab (Ipi) improves the survival of advanced melanoma patients with an incremental long-term benefit in 10–15 % of patients. A tumor signature that correlates with this survival benefit could help optimizing individualized treatment strategies.

The treatment of locally advanced metastasized melanoma is challenging because there is no level 1 evidence to guide clinical decision-making. Moreover, the treatment options available fail to improve overall survival and are associated with considerable morbidity. Here, we show that systemic treatment with BRAF inhibitor vemurafenib substituted by dual BRAF/MEK inhibition (dabrafenib and trametinib) before surgery can offer the potential to cure the initially difficult or inoperable melanoma. A 62-year-old woman was diagnosed with an AJCC stage IIIB melanoma harboring the BRAF V600E mutation after a complete initial evaluation. Clinically, the patient presented a large primary lesion that was surrounded by ∼25 secondary epidermotropic lesions both satellite and 'in transit' with a diameter between 1 and 6 mm. Following multidisciplinary consultation, the patient was started on 960 mg twice-daily vemurafenib, which was stopped and resumed at 720 mg twice daily, and finally substituted with the combination dabrafenib and trametinib to reduce the persistent side effects. Successive clinical examinations had shown a progressive reduction in the thickness of the melanoma lesions. After about 5 months of therapy, surgery was performed and the histopathological analysis showed an almost complete regression of tumor cells. The treatment with dabrafenib/trametinib was continued only 3 months after surgery and stopped at the patient's request. The patient currently remains in complete remission at 8 months after surgery. The case presented here supports the use of neoadjuvant treatment with BRAF inhibitors in advanced 'in transit' melanoma.

9052 Background: Ipilimumab (Ipi) improves the survival of patients (pts) with advanced melanoma. Combination of Ipi with an autologous monocyte-derived DC therapy (TriMixDC-MEL) may further improve patient outcome (Neyns et al., 2014 ASCO AM). A predictive melanoma tissue signature for the clinical efficacy of Ipi and TriMixDC is needed to optimize individualized treatment strategies. Methods: Between 01/2011 and 05/2013 freshly frozen melanoma metastases were collected from pts treated with Ipi (n: 9), TriMixDC-MEL (n: 2), or TriMixDC-MEL plus Ipi (n: 14). Samples were profiled by IHC (incl. CD3, CD8, PanMel, MCSP, CD20, CD163, DC-LAMP, Casp-3, Ki-67, PHH3, HLA class I, vWF), RNA-seq and MBD-seq (genome-wide DNA methylation profiling). Tumor response was assessed according to the irRC. Results: Five pts obtained a complete response (CR), 6 obtained a mixed response (MR), and there was no evidence of tumor response in 14 pts (NR). A total of 26 metastases (15 obtained prior to and 11 post therapy) were profiled by IHC and 18 were additionally profiled by RNA and MBD-seq. Intra-tumor CD3 and CD8 T cell infiltration was observed in 23/26 samples, with variable pattern and extent of infiltration. None of the 3 pts with absent CD3/CD8 infiltration in their tumor core regions responded to Ipi-based immunotherapy. The differential analysis of the RNA-seq data resulted in a list of 195 genes with a statistically significant difference in expression between CR and NR (false discovery rate < 0.05). A gene ontology enrichment analysis revealed that the differentially expressed genes were enriched for immune-related ontologies. The MR expression profiles clustered together with the NR group. MBD-seq revealed differences in methylation status between CR and NR pts. These differences in methylation status were not linked to the observed differences in RNA expression. Conclusions: Comprehensive analysis of melanoma metastases with gene expression and methylation status provides a distinct profile that identifies long-term responders to Ipi-based immunotherapy. Prospective validation is warranted.

The aim of this study was to determine the activity of ipilimumab (ipi)-based therapy after treatment failure with a BRAF inhibitor (BRAFi). Sixty-four patients with unresectable stage III or stage IV BRAF V600-mutant melanoma who were treated sequentially with a BRAFi and ipi-based therapy [ipi as monotherapy or ipi in combination with an autologous mRNA electroporated dendritic cell vaccine (TriMixDC-MEL)] were identified. Thirty-three patients had been treated with a BRAFi before ipi-based therapy (BRAFi-first), and 31 patients had been treated with ipi-based therapy first (ipi-first). In patients treated with a BRAFi first (n=33), the best response on sequential ipi-based therapy was three complete responses and six partial responses (best objective response rate of 27%). In patients treated with ipi-based therapy first (n=31), the best response on ipi-based therapy was 0 complete response and four partial responses (best objective response rate of 13%). The response rate did not differ significantly between the two groups (P=0.14). The median overall survival from the start of ipi-based therapy was 10 months (95% confidence interval: 5.7–14.3) in the BRAFi-first group and 12.3 months (95% confidence interval: 7.4–17.2) in the ipi-first group (P=0.34). We report that objective tumor responses to ipi-based immunotherapy can still be obtained after progression has occurred upon treatment with a BRAFi. A part of this observation might be related to the results obtained with a combination of ipi and TriMixDC-MEL.

Ipilimumab, a CTLA-4-blocking monoclonal antibody, improved the overall survival (OS) of advanced melanoma patients treated in prospective clinical trials. We here report a study on the outcome of patients with pretreated advanced melanoma offered ipilimumab (at its licensed dose of 3 mg/kg, every 3 wk for a total of 4 doses) in an expanded access program at a single-center university hospital. Of the 50 patients initiating ipilimumab, 31 patients completed induction therapy and 9 patients were offered reinduction therapy. Most immune-related adverse events were mild and reversible. The best objective response rate by mWHO-criteria included 1 complete response and 4 partial responses (best objective response rate of 10%). Two additional patients obtained a partial response by immune-related response criteria. Median OS was 7 months, with a 1- and 2-year survival rate of 45.2% and 28.8%, respectively. Long-term disease control with ipilimumab was observed in 7 patients of which 4 received reinduction. Baseline serum C-reactive protein (CRP) and the absolute lymphocyte count (ALC) measured on week 6 significantly correlated with OS. In conclusion, in this single-center experience with ipilimumab for advanced pretreated melanoma patients, clinical outcome was comparable with the results of published prospective studies. Reinduction therapy was of importance for maintaining long-term disease control in the majority of responding patients. Baseline CRP and ALC at week 6 deserve further prospective evaluation as prognostic and/or predictive (surrogate) markers.

Summary Recombinant tumour necrosis factor-α (rTNFα) possesses the unique property of activating and selectively destroying the tumour-associated microvasculature. Systemic application of rTNFα has shown that the maximum tolerated dose (MTD) is 10 times lower than the efficient dose in animals. The main toxicity corresponds to the systemic inflammatory response syndrome (SIRS), with a decrease of vascular resistance and hypotension. We found that it is possible to administer rTNFα at 10 times the MTD in an isolated limb perfusion system, using a heart-lung machine, for advanced melanoma and sarcoma of the limbs. Our results, using the combination of high dose rTNFα, interferon-γ and melphalan (TIM), produced an overall objective response rate of 100% in 2 successive studies on melanoma, with 90% and 78% complete response, respectively. In sarcoma, there was an overall response rate of 64%, with 36% complete response. Angiographic and immunohistological studies demonstrated selective and early damage of the tumour-associated microvasculature, preceded by upregulation of adhesion molecules and intratumoural leak of von Willebrand factor. Tumour invasion by platelets and, in some cases, by polymorphonuclear cells, appeared within hours after the application of rTNFα, long before the lysis of the tumour. Systemic changes after rTNFα treatment included the production of soluble TNFα receptors and of interleukin-6. A typical acute phase reaction was observed within 3 days, with increase of C-reactive protein parallelled by an increase of tenascin-C. A selective effect on intratumoural endothelial cells seems to be involved in the mechanism of the impressive antitumour effect of rTNFα, but the role of acute phase protein production is not fully understood. In selected cases of melanoma, specific cytotoxic T lymphocytes were increased after perfusion.

e19026 Background: Ipilimumab (IPI) is a CTLA-4 blocking mAb with established activity in patients (pts) with advanced melanoma. Methods: Observational study on the outcome of advanced melanoma pts treated with IPI in an expanded access program (EAP) at a single-center. Results: Fifty pts initiated IPI (3 mg/kg q3wks x4, allowing for reinduction after a PFS of >24 wks). Baseline characteristics: 28M/22F, m50y, Stage: 1x IV-M1a, 2x -M1b, 47x -M1c; 44 pts skin-, 2x mucosal-, and 4x unknown primary melanoma; WHO-PS 0/1/2: 17-, 25-, 8 pts; LDH >ULN 33 pts; CRP >ULN 29 pts; ALC<1000/mm³ 15 pts; all pts were pretreated with dacarbazine and 16 pts for brain metastases. Thirty-one pts completed IPI-induction; 8/11 pts offered reinduction completed the planned 4 administrations (treatment ongoing in 1pt). Most immune-related adverse events (irAE) were generally mild/reversible; CTCAE gr3 irAE: diarrhea/colitis (3), hepatitis (1), hypophysitis (1), pancreatitis (1) and Guillain-Barré syndrome (1); no grade 4/5 irAE. BORR by mWHO-criteria: 1 CR, 3 PR and 4 SD (disease control rate [DCR]: 16%). BORR by immune-related response criteria (irRC): additional 2 PR and 3 SD (DCR: 26%). In 5/10 evaluable pts a similar or lower total tumor burden was obtained following reinduction. Two pts who experienced gr3 irAE during induction did not experience the same irAE at reinduction. After a median follow-up of 18 mths (range 7.5-19.3), 32 pts have died. Six- and 12-mths OS were 53% (95% CI 40-67) and 45% (95%CI 31-59) respectively. Disease control with IPI has been maintained in 8 pts (of which 4 received reinduction) after a median follow-up of 17 mths (range 12-17). Elevated CRP (>5x the upper limit of normal [ULN]) was the only independent baseline co-variable (HR 0,10; 95% CI 0,04-0,25; p 0.001). An ALC of <800/mm³ at week 7 and disease-control by irRC post-induction significantly correlated with OS. Conclusions: This single-center experience with IPI in an EAP for pts with advanced pretreated melanoma is comparable with that of prospective studies. Reinduction therapy seems important for long-term disease control in responding pts. Baseline CRP and ALC at week 7 deserve further evaluation as a prognostic and/or predictive (surrogate) markers.

S100B is a prognostic factor for melanoma as elevated levels correlate with disease progression and poor outcome. We determined its prognostic value based on updated information using serial determinations in stage IIb/III melanoma patients. 211 Patients who participated in the EORTC 18952 trial, evaluating efficacy of adjuvant intermediate doses of interferon α2b (IFN) versus observation, entered a corollary study. Over a period of 36 months, 918 serum samples were collected. The Cox time-dependent model was used to assess prognostic value of the latest (most recent) S100B determination. At first measurement, 178 patients had S100B values <0.2 μg/l and 33 ≥ 0.2 μg/l. Within the first group, 61 patients had, later on, an increased value of S100B (≥ 0.2 μg/l). An initial increased value of S100B, or during follow-up, was associated with worse distant metastasis-free survival (DMFS); hazard ratio (HR) of S100B ≥ 0.2 versus S100B < 0.2 was 5.57 (95% confidence interval (CI) 3.81-8.16), P < 0.0001, after adjustment for stage, number of lymph nodes and sex. In stage IIb patients, the HR adjusted for sex was 2.14 (95% CI 0.71, 6.42), whereas in stage III, the HR adjusted for stage, number of lymph nodes and sex was 6.76 (95% CI 4.50-10.16). Similar results were observed regarding overall survival (OS). Serial determination of S100B in stage IIb-III melanoma is a strong independent prognostic marker, even stronger compared to stage and number of positive lymph nodes. The prognostic impact of S100B ≥ 0.2 μg/l is more pronounced in stage III disease compared with stage IIb.

Cancer-testis (CT) antigens comprise families of tumor-associated antigens that are immunogenic in patients with various cancers. Their restricted expression makes them attractive targets for immunotherapy. The aim of this study was to determine the expression of several CT genes and evaluate their prognostic value in head and neck squamous cell carcinoma (HNSCC). The pattern and level of expression of 12 CT genes (MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A10, MAGE-C2, NY-ESO-1, LAGE-1, SSX-2, SSX-4, BAGE, GAGE-1/2, GAGE-3/4) and the tumor-associated antigen encoding genes PRAME, HERV-K-MEL, and NA-17A were evaluated by RT-PCR in a panel of 57 primary HNSCC. Over 80% of the tumors expressed at least 1 CT gene. Coexpression of three or more genes was detected in 59% of the patients. MAGE-A4 (60%), MAGE-A3 (51%), PRAME (49%) and HERV-K-MEL (42%) were the most frequently expressed genes. Overall, the pattern of expression of CT genes indicated a coordinate regulation; however there was no correlation between expression of MAGE-A3/A4 and BORIS, a gene whose product has been implicated in CT gene activation. The presence of MAGE-A and NY-ESO-1 proteins was verified by immunohistochemistry. Analysis of the correlation between mRNA expression of CT genes with clinico-pathological characteristics and clinical outcome revealed that patients with tumors positive for MAGE-A4 or multiple CT gene expression had a poorer overall survival. Furthermore, MAGE-A4 mRNA positivity was prognostic of poor outcome independent of clinical parameters. These findings indicate that expression of CT genes is associated with a more malignant phenotype and suggest their usefulness as prognostic markers in HNSCC.

PURPOSE:As no curative treatment for advanced pancreatic and biliary cancer with malignant ascites exists, new modalities possibly improving the response to available chemotherapies must be explored. This phase I study assesses the feasibility, tolerability and pharmacokinetics of a regional treatment of gemcitabine administered in escalating doses by the stop-flow approach to patients with advanced abdominal malignancies (adenocarcinoma of the pancreas, n = 8, and cholangiocarcinoma of the liver, n = 1). EXPERIMENTAL DESIGN:Gemcitabine at 500, 750 and 1,125 mg/m(2) was administered to three patients at each dose level by loco-regional chemotherapy, using hypoxic abdominal stop-flow perfusion. This was achieved by an aorto-caval occlusion by balloon catheters connected to an extracorporeal circuit. Gemcitabine and its main metabolite 2',2'-difluorodeoxyuridine (dFdU) concentrations were measured by high performance liquid chromatography with UV detection in the extracorporeal circuit during the 20 min of stop-flow perfusion, and in peripheral plasma for 420 min. Blood gases were monitored during the stop-flow perfusion and hypoxia was considered stringent if two of the following endpoints were met: pH = 7.2, pO(2) nadir ratio =0.70 or pCO(2) peak ratio >/=1.35. The tolerability of this procedure was also assessed. RESULTS:Stringent hypoxia was achieved in four patients. Very high levels of gemcitabine were rapidly reached in the extracorporeal circuit during the 20 min of stop-flow perfusion, with C (max) levels in the abdominal circuit of 246 (+/-37%), 2,039 (+/-77%) and 4,780 (+/-7.3%) mug/ml for the three dose levels 500, 750 and 1,125 mg/m(2), respectively. These C (max) were between 13 (+/-51%) and 290 (+/-12%) times higher than those measured in the peripheral plasma. Similarly, the abdominal exposure to gemcitabine, calculated as AUC(t0-20), was between 5.5 (+/-43%) and 200 (+/-66%)-fold higher than the systemic exposure. Loco-regional exposure to gemcitabine was statistically higher in presence of stringent hypoxia (P < 0.01 for C (max) and AUC(t0-20), both normalised to the gemcitabine dose). Toxicities were acceptable considering the complexity of the procedure and were mostly hepatic; it was not possible to differentiate the respective contributions of systemic and regional exposures. A significant correlation (P < 0.05) was found between systemic C (max) of gemcitabine and the nadir of both leucocytes and neutrophils. CONCLUSIONS:Regional exposure to gemcitabine-the current standard drug for advanced adenocarcinoma of the pancreas-can be markedly enhanced using an optimised hypoxic stop-flow perfusion technique, with acceptable toxicities up to a dose of 1,125 mg/m(2). However, the activity of gemcitabine under hypoxic conditions is not as firmly established as that of other drugs such as mitomycin C, melphalan or tirapazamine. Further studies of this investigational modality, but with bioreductive drugs, are therefore warranted first to evaluate the tolerance in a phase I study and later on to assess whether it does improve the response to chemotherapy.

Toll-like receptor ligands are potentially useful adjuvants for the development of clinical T cell vaccination. Here we investigated the novel Toll-like receptor2 ligand P40, the outer membrane protein A derived from Klebsiella pneumoniae. Seventeen human leukocyte antigen-A*0201 positive stage III/IV melanoma patients were vaccinated with P40 and Melan-A/Mart-1 peptide subcutaneously in monthly intervals. Adverse reactions were mild-to-moderate. Fourteen patients received at least 8 vaccinations and were thus evaluable for clinical tumor and immune responses. Seven patients experienced progressive disease, whereas 2 patients had stable disease throughout the trial period, 1 of them with regression of multiple skin metastases. The remaining 5 patients had no measurable disease. Melan-A/Mart-1 specific CD8+ T cells were analyzed ex vivo, with positive results in 6 of 14 evaluable patients. Increased percentages of T cells were found in three patients, memory/effector T cell differentiation in 4 patients, and a positive interferon-γ Elispot assay in 1 patient. Antibody responses to P40 were observed in all patients. We conclude that vaccination with peptide and P40 was feasible and induced ex vivo detectable tumor antigen specific T cell responses in 6 of 14 patients with advanced melanoma.

Galectin-3 (Gal-3) is a member of the P-galactoside-binding lectins family and has been implicated in angiogenesis, tumor invasion, and metastatic process in vitro and in vivo. As we showed recently that advanced melanoma patients presented high serum level of Gal-3, we investigated the association of this protein with the outcome of melanoma patients. Whether this protein could be a biomarker has riot been assessed, and we compared the prognostic value of serum Gal-3 in multivariate analysis with lactate dehydrogenase, C-reactive protein and S100B. We conclude that Gal-3 could be of prognostic value in melanoma patients; more precisely, this protein has a strong independent prognostic signification with a cut-off value of 10 ng/ml. After these data, we believe that serum Gal-3 measurement can have an important role in the follow-up and management of advanced American Joint Commission on Cancer stage III and stage IV melanoma patients. Further studies will uncover whether Gal-3 will be able to open new therapeutic perspectives. Melanoma Res 19:316-320 (C) 2009 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Activated CD8 T cells develop cytotoxicity against autologous cells bearing foreign Ags and self/tumor Ags. However, self-specific cytolysis needs to be kept under control to avoid overwhelming immunopathology. After peptide vaccination of melanoma patients, we studied molecular and functional properties of T cell subsets specific for the self/tumor Ag Melan-A/MART-1. Ex vivo analysis revealed three Ag-specific effector memory (EM) populations, as follows: CD28-negative EM (EM28(-)) T cells strongly expressing granzyme/perforin, and two EM28(+) subsets, one with high and the other with low level expression of these cytotoxic proteins. For further functional characterization, we generated 117 stable CD8 T cell clones by ex vivo flow cytometry-based sorting of these subsets. All EM28(-)-derived clones lysed target cells with high efficacy. In contrast, EM28(+)-derived clones were heterogenous, and could be classified in two groups, one with high and the other with low killing capacity, correlating with granzyme/perforin expression. High and low killer phenotypes remained surprisingly stable for several months. However, strongly increased granzyme expression and cytotoxicity were observed after exposure to IL-12. Thus, the data reveal a newly identified subset of CD28(+) conditional killer T cells. Because CD28 can mediate strong costimulatory signals, tight cytotoxicity control, as shown in this study through IL-12, may be particularly important for subsets of T cells expressing CD28.

Aim: To confirm the accuracy of sentinel node biopsy (SNB) procedure and its morbidity, and to investigate predictive factors for SN status and prognostic factors for disease-free survival (DFS) and disease-specific survival (DSS).Materials and methods: Between October 1997 and December 2004, 327 consecutive patients in one centre with clinically node-negative primary skin melanoma underwent an SNB by the triple technique, i.e. lymphoscintigraphy, blue-dye and gamma-probe. Multivariate logistic regression analyses as well as the Kaplan-Meier were performed.Results: Twenty-three percent of the patients had at least one metastatic SN, which was significantly associated with Breslow thickness (p < 0.001). The success rate of SNB was 99.1 % and its morbidity was 7.6%. With a median follow-up of 33 months, the 5-year DFS/DSS were 43%/49% for patients with positive SN and 83.5%/87.4% for patients with negative SN, respectively. The false-negative rate of SNB was 8.6% and sensitivity 91.4%. On multivariate analysis, DFS was significantly worsened by Breslow thickness (RR = 5.6, p < 0.001), positive SN (RR = 5.0, p < 0.001) and male sex (RR = 2.9, p = 0.001). The presence of a metastatic SN (RR = 8.4, p < 0.001), male sex (RR = 6.1, p < 0.001), Breslow thickness (RR = 3.2, p = 0.013) and ulceration (RR = 2.6, p = 0.015) were significantly associated with a poorer DSS.Conclusion: SNB is a reliable procedure with high sensitivity (91.4%) and low morbidity. Breslow thickness was the only statistically significant parameter predictive of SN status. DFS was worsened in decreasing order by Breslow thickness, metastatic SN and male gender. Similarly DSS was significantly worsened by a metastatic SN, male gender, Breslow thickness and ulceration. These data reinforce the SN status as a powerful staging procedure. (c) 2007 Elsevier Ltd. All rights reserved.