Natural killer T (NKT) cells are CD1d-restricted lymphoid cells and are characterized by an invariant T-cell receptor, which in humans consists of a Va24 chain paired with a Vb11 chain. These cells are known for their rapid production of large amounts of cytokines (e.g., Vb11+ NKT cell numbers in both healthy controls and cancer patients and demonstrate that in both groups females have higher NKT cell levels compared to males. In a large group of 120 cancer patients, we show that circulating Va24+ NKT cell numbers are about 50% lower than in ageand gender-matched healthy controls and that this decrease is independent of tumor type or tumor load. This decrease was not restored upon tumor removal by means of surgery or radiotherapy. Even though the percentage of NKT cells that secrete IFN-g, as detected by ELISPOT, is normal in cancer patients, the absolute number of circulating IFN-g-secreting NKT cells is reduced. Together, our results suggest that the reduced circulating Va24 + Vb11+ NKT cell numbers in cancer patients are not affected by tumor load, but might actually reflect a risk factor for tumor development, e.g., by hampering efficient tumor immunosurveillance. ' 2005 Wiley-Liss, Inc.

Purpose: A decrease in the frequency and activation state of dendritic cells in the sentinel lymph node (SLN) has been observed in early stages of melanoma development. This may hinder the generation of effective antitumorT-cell responses and increase the likelihood of metastatic spread. Immunopotentiation of the melanoma SLN may therefore be a valuable adjuvant treatment option. One way to achieve this is through the use of bacterially derived unmethylated cytosinephosphate-guanine (CpG) DNA sequences that bind Toll-like receptor 9 and activate plasmacytoid dendritic cells (PDC). CpG-activated PDC, in turn, release IFNa and may thus boost T-cell and natural killer cell responses as well as activate conventional myeloid dendritic cells (MDC).

Purpose: Impaired immune effector functions in the melanoma sentinel lymph node (SLN) may allow for early metastatic events. Local administration of PF-3512676 (formerly known as CpG 7909) has shown immunostimulatory effects of both dendritic cell and T-cell subsets in the melanoma SLN. Here, we set out to ascertain whether these PF-3512676-induced immunostimulatory effects translate into higher frequencies of melanoma-specific CD8 + Tcells. Experimental Design: Twenty-four stage I to III melanoma patients were randomized to preoperative local administration of either PF-3512676 or saline. CD8 + T cells from SLN and peripheral blood were tested for reactivity by IFN-g ELISPOT assay against several HLA-A1/A2/ A3-restricted epitopes derived from various melanoma-associated antigens (MAA) in 21 of 24 enrolled patients. Frequencies of natural killer (NK) cells and frequencies and maturation state of dendritic cell subsets in the SLN were determined by flow cytometry. Results: Melanoma-specific CD8 + T-cell response rates against >1MAA epitope in the SLN were 0 of 11 for the saline group versus 5 of 10 for the PF-3512676-administered group (P = 0.012).Of these 5 responding patients, 4 also had a measurable response to >1 MAA epitope in the blood. Increased frequencies in the SLN of both MAA-specific CD8 + T cells and NK cells correlated to CpG-induced plasmacytoid dendritic cell maturation. Conclusions: These data show an increase in melanoma-specific CD8 + T-cell frequencies as well as an increased effector NK cell rate after a single dose of PF-3512676 and thus support the utility of local PF-3512676 administration as adjuvant treatment in early-stage melanoma to try and halt metastatic spread.

In this study we investigated whether the presence of specific populations of tumor infiltrating lymphocytes (TILs) in diagnostic primary melanoma biopsies are related to outcome in clinically stage II melanoma patients. Moreover, we investigated whether the presence of TILs correlates with expression of MHC class I antigen and MHC class II antigen on tumor cells and/or tumor infiltrating antigen presenting cells. Diagnostic primary melanoma samples of 15 patients with an unfavorable outcome were compared with 20 patients with favorable outcome. Patients were matched for age, gender and Breslow thickness. Biopsies were examined for the presence of granzyme B 1 , CD8 1 , CD4 1 and CD56 1 TILs and for expression of MHC class I antigen and MHC class II antigen on tumor and/or tumor infiltrating cells. A favorable clinical outcome was strongly associated with the presence of GrB 1 and CD4 1 TILs, with expression of MHC class I antigen on tumor cells and with expression of MHC class II antigen on intratumoral antigen presenting cells. These data strongly support the notion that in melanoma patients the cellular immune response is a major factor in preventing melanoma cell dissemination. ' 2008 Wiley-Liss, Inc.Key words: TILs; granzyme B; MHC class I; MHC class II; prognosis; CD4; CD8 Even though melanomas account for only 4% of all skin cancers, they cause the greatest number of skin cancer-related deaths worldwide. Over the last few decades an increase in incidence and mortality has been observed in Caucasian populations across the world. 1,2 Clinical outcome in melanoma patients depends on several variables of which tumor thickness is an important factor (according to Breslow). 3 The 5 year survival rate for patients with a Breslow thickness <1.5 mm is more than 90%, whereas survival in patients with a Breslow thickness of >3.5 mm is only 50%. 4 Other important prognostic factors are, amongst others, gender and age. [5][6][7] Fatal outcome in melanoma patients often results from occurrence of distant metastases, which mostly coincide or are preceded by lymph node metastases. In line with this concept, previous studies demonstrated that patients with a melanoma sentinel lymph node (SLN) metastasis have a worse prognosis than patients without a SLN metastasis. 8,9 However, despite known prognostic parameters, outcome often remains unpredictable and further research to identify additional relevant prognostic markers is warranted.It has previously been shown that melanomas can elicit an immune response 10,11 and that melanoma cells can effectively be eradicated in vivo by cytotoxic activity of MHC class I antigen restricted CD8 1 Granzyme B (GrB 1 ) T-cells. 12 Thus, a possible explanation for differences in clinical outcome might be that a proper immune response, although incapable of preventing the primary tumor from growing, is able to prevent the occurrence of lymph node and/or distant metastases. A large number of studies have shown that the cellular immune response plays an important role in the control of melan...

Recognition of the tumor during breast-conserving surgery (BCS) can be very difficult and currently a robust method of margin assessment for the surgical setting is not available. As a result, tumor-positive margins, which require additional treatment, are not found until histopathologic evaluation. With diffuse reflectance spectroscopy (DRS), tissue can be characterized during surgery based on optical parameters that are related to the tissue morphology and composition. Here we investigate which optical parameters are able to detect tumor in an area with a mixture of benign and tumor tissue and hence which parameters are most suitable for intra-operative margin assessment. DRS spectra (400-1600 nm) were obtained from 16 ex vivo lumpectomy specimens from benign, tumor border, and tumor tissue. One mastectomy specimen was used with a custom-made grid for validation purposes. The optical parameter related to the absorption of fat and water (F/W-ratio) in the extended near-infrared wavelength region (~1000-1600 nm) provided the best discrimination between benign and tumor sites resulting in a sensitivity and specificity of 100 % (excluding the border sites). Per patient, the scaled F/W-ratio gradually decreased from grossly benign tissue towards the tumor in 87.5 % of the specimens. In one test case, based on a predefined F/W-ratio for boundary tissue of 0.58, DRS produced a surgical resection plane that nearly overlapped with a 2-mm rim of benign tissue, 2 mm being the most widely accepted definition of a negative margin. The F/W-ratio provided excellent discrimination between sites clearly inside or outside the tumor and was able to detect the border of the tumor in one test case. This work shows the potential for DRS to guide the surgeon during BCS.