Lentivirus-induced 'Smart' dendritic cells: Pharmacodynamics and GMP-compliant production for immunotherapy against TRP2-positive melanoma.

Monocyte-derived conventional dendritic cells (ConvDCs) loaded with melanoma antigens showed modest responses in clinical trials. Efficacy studies were hampered by difficulties in ConvDC manufacturing and low potency. Overcoming these issues, we demonstrated higher potency of lentiviral vector (LV)-programmed DCs. Monocytes were directly induced to self-differentiate into DCs (SmartDC-TRP2) upon transduction with a tricistronic LV encoding for cytokines (granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4)) and a melanoma antigen (tyrosinase-related protein 2 (TRP2)). Here, SmartDC-TRP2 generated with monocytes from five advanced melanoma patients were tested in autologous DC:T cell stimulation assays, validating the activation of functional TRP2-specific cytotoxic T lymphocytes (CTLs) for all patients. We described methods compliant to good manufacturing practices (GMP) to produce LV and SmartDC-TRP2. Feasibility of monocyte transduction in a bag system and cryopreservation following a 24-h standard operating procedure were achieved. After thawing, 50% of the initial monocyte input was recovered and SmartDC-TRP2 self-differentiated in vitro, showing uniform expression of DC markers, detectable LV copies and a polyclonal LV integration pattern not biased to oncogenic loci. GMP-grade SmartDC-TRP2 expanded TRP2-specific autologous CTLs in vitro. These results demonstrated a simpler GMP-compliant method of manufacturing an effective individualized DC vaccine. Such DC vaccine, when in combination with checkpoint inhibition therapies, might provide higher specificity against melanoma.

Epigenetic status of LINE-1 predicts clinical outcome in early-stage rectal cancer.

BACKGROUND: We evaluated the clinical prognostic value of methylation of two non-coding repeat sequences, long interspersed element 1 (LINE-1) and Alu, in rectal tumour tissues. In addition to DNA methylation, expression of histone modifications H3K27me3 and H3K9Ac was studied in this patient cohort. METHODS: LINE-1 and Alu methylation were assessed in DNA extracted from formalin-fixed paraffin-embedded tissues. A pilot (30 tumour and 25 normal tissues) and validation study (189 tumour and 53 normal tissues) were performed. Histone modifications H3K27me3 and H3K9Ac were immunohistochemically stained on tissue microarrays of the study cohort. RESULTS: In early-stage rectal cancer (stage I-II), hypomethylation of LINE-1 was an independent clinical prognostic factor, showing shorter patient survival (P=0.014; HR: 4.6) and a higher chance of tumour recurrence (P=0.001; HR: 9.6). Alu methylation did not show any significant correlation with clinical parameters, suggesting an active role of LINE-1 in tumour development. Expression of H3K27me3 (silencing gene expression) and H3K9Ac (activating gene expression) in relation to methylation status of LINE-1 and Alu supported this specific role of LINE-1 methylation. CONCLUSION: The epigenetic status of LINE-1, but not of Alu, is prognostic in rectal cancer, indicating an active role for LINE-1 in determining clinical outcome.

Assessment of DNA methylation status in early stages of breast cancer development.

BACKGROUND: Molecular pathways determining the malignant potential of premalignant breast lesions remain unknown. In this study, alterations in DNA methylation levels were monitored during benign, premalignant and malignant stages of ductal breast cancer development. METHODS: To study epigenetic events during breast cancer development, four genomic biomarkers (Methylated-IN-Tumour (MINT)17, MINT31, RARß2 and RASSF1A) shown to represent DNA hypermethylation in tumours were selected. Laser capture microdissection was employed to isolate DNA from breast lesions, including normal breast epithelia (n=52), ductal hyperplasia (n=23), atypical ductal hyperplasia (n=31), ductal carcinoma in situ (DCIS, n=95) and AJCC stage I invasive ductal carcinoma (IDC, n=34). Methylation Index (MI) for each biomarker was calculated based on methylated and unmethylated copy numbers measured by Absolute Quantitative Assessment Of Methylated Alleles (AQAMA). Trends in MI by developmental stage were analysed. RESULTS: Methylation levels increased significantly during the progressive stages of breast cancer development; P-values are 0.0012, 0.0003, 0.012, <0.0001 and <0.0001 for MINT17, MINT31, RARß2, RASSF1A and combined biomarkers, respectively. In both DCIS and IDC, hypermethylation was associated with unfavourable characteristics. CONCLUSION: DNA hypermethylation of selected biomarkers occurs early in breast cancer development, and may present a predictor of malignant potential.

Aberrant hypermethylation in primary tumours and sentinel lymph node metastases in paediatric patients with cutaneous melanoma.

BACKGROUND: Debate on how to manage paediatric patients with cutaneous melanoma continues, particularly in those with sentinel lymph node (SLN) metastases who are at higher risk of poor outcomes. Management is often based on adult algorithms, although differences in clinical outcomes between paediatric and adult patients suggest that melanoma in paediatric patients differs biologically. Yet, there are no molecular prognostic studies identifying these differences. OBJECTIVES: We investigated the epigenetic (methylation) regulation of several tumour-related genes (TRGs) known to be significant in adult melanoma progression in histopathology(+) SLN metastases (n = 17) and primary tumours (n = 20) of paediatric patients with melanoma to determine their clinical relevance. METHODS: Paediatric patients (n = 37; ≤ 21 years at diagnosis) with American Joint Committee on Cancer stage I-III cutaneous melanoma were analysed. Gene promoter methylation of the TRGs RASSF1A, RARß2, WIF1 and APC was evaluated. RESULTS: Hypermethylation of RASSF1A, RARß2, WIF1 and APC was found in 29% (5/17), 25% (4/16), 25% (4/16) and 19% (3/16) of histopathology(+) SLNs, respectively. When matched to adult cutaneous melanomas by Breslow thickness and ulceration, hypermethylation of all four TRGs in SLN(+) paediatric patients with melanoma was equivalent to or less than in adults. With a median follow-up of 55 months, SLN(+) paediatric patients with melanoma with hypermethylation of > 1 TRG vs. ≤ 1 TRG had worse disease-free (P = 0·02) and overall survival (P = 0·02). CONCLUSIONS: Differences in the methylation status of these TRGs in SLN(+) paediatric and adult patients with melanoma may account for why SLN(+) paediatric patients have different clinical outcomes. SLN biopsy should continue to be performed; within SLN(+) paediatric patients with melanoma, hypermethylation of TRGs can be used to identify a subpopulation at highest risk for poor outcomes who warrant vigilant clinical follow-up.

Immunity against breast cancer by TERT DNA vaccine primed with chemokine CCL21.

Human telomerase reverse transcriptase (TERT) has been considered a potential tumor-associated antigen for active-specific immunotherapy. However, effective specific tumor antigen-specific immunity has been difficult to induce consistently by various TERT vaccine formulations. New adjuvant strategies have been employed, such as utilizing chemokines to attract T cells and antigen-presenting cells. Chemokine adjuvant strategies may enhance tumor antigen-specific immunity induced by vaccines. Therefore, we utilized chemokine ligand 21 (CCL21) as an adjuvant with a xenogeneic TERT DNA vaccine to induce tumor antigen-specific immunity against TERT-expressing breast cancer. The TERT DNA vaccine consisted of a plasmid containing the COOH terminal end of the TERT (cTERT) gene, encapsulated in multilayered liposomes with hemagglutinating virus of Japan coating. We demonstrated that CCL21 treatment before cTERT DNA vaccine, given intramuscularly, induced significantly higher anti-TERT specific cell-mediated immunity compared to cTERT DNA vaccine alone. Effective tumor antigen-specific immunity was shown both in prophylactic and therapeutic regimens against TS/A murine breast cancer. The study demonstrated that CCL21 administration before cTERT DNA vaccination significantly augmented tumor antigen-specific immunity against breast cancer.

Unfavourable prognosis associated with K-ras gene mutation in pancreatic cancer surgical margins.

BACKGROUND: Despite intent to cure surgery with negative resection margins, locoregional recurrence is common in pancreatic cancer. AIMS: To determine whether detection of K-ras gene mutation in the histologically negative surgical margins of pancreatic cancer reflects unrecognised disease. PATIENTS: Seventy patients who underwent curative resection for pancreatic ductal adenocarcinoma were evaluated. METHODS: All patients had surgical resection margins (pancreatic transection and retroperitoneal) that were histologically free of invasive cancer. DNA was extracted from these paraffin embedded surgical margins and assessed by quantitative real time polymerase chain reaction to detect the K-ras gene mutation at codon 12. Detection of K-ras mutation was correlated with standard clinicopathological factors. RESULTS: K-ras mutation was detected in histologically negative surgical margins of 37 of 70 (53%) patients. A significant difference in overall survival was demonstrated between patients with margins that were K-ras mutation positive compared with negative (median 15 v 55 months, respectively; p = 0.0008). By univariate and multivariate analyses, detection of K-ras mutation in the margins was a significant prognostic factor for poor survival (hazard ratio (HR) 2.8 (95% confidence interval (CI) 1.5-5.3), p = 0.0009; and HR 2.8 (95% CI 1.4-5.5), p = 0.004, respectively). CONCLUSIONS: Detection of cells harbouring K-ras mutation in histologically negative surgical margins of pancreatic cancer may represent unrecognised disease and correlates with poor disease outcome. The study demonstrates that molecular-genetic evaluation of surgical resection margins can improve pathological staging and prognostic evaluation of patients with pancreatic ductal adenocarcinoma.

Ultrastaging of early colon cancer using lymphatic mapping and molecular analysis.

Approximately one-third of node-negative colon cancers will recur, possibly due to understaging and inadequate pathological examination of lymph nodes (LNs). We evaluated the sensitivity, accuracy and feasibility of staging based on lymphatic mapping, focused examination, and molecular analysis of the sentinel node (SN) in patients with primary colorectal carcinoma. Between 1996 and 2000, 100 patients with colon carcinoma (CRC) underwent lymphatic mapping immediately after peritumoral injection of 1.0 cc of isosulphan blue dye. All LNs in the CRC specimen were examined by routine haematoxylin and eosin (H&E) staining. Sentinel nodes were examined by step serial sectioning, cytokeratin immunohistochemistry (CK-IHC) and/or reverse transcriptase-polymerase chain reaction (RT-PCR) analysis in an attempt to identify occult micrometastatic disease. Lymphatic mapping was successful in 97% of the cases. There were 5 false-negative cases, predominately associated with T3/T4 tumours. Aberrant lymphatic drainage was identified in 8 patients (8%) altering the operative approach. 26 patients had H&E-positive LNs. In 74 patients who were node-negative by routine H&E, 18 (24%) had occult nodal micrometastases missed on routine H&E examination, but detected by focused analysis of the SN. RT-PCR analysis of the SN was performed in 40 patients, 26 of which were negative by H&E and CK-IHC. In 12/26 (46%) of these patients, there was additional evidence of micrometastatic disease. In this study, focused examination of the SN in conjunction with RT-PCR analysis identified micrometastatic disease in a significant number of node-negative patients. This may have important implications when selecting patients for adjuvant treatment protocols.