A practical approach to RT-qPCR-Publishing data that conform to the MIQE guidelines.

Given the highly dynamic nature of mRNA transcription and the potential variables introduced in sample handling and in the downstream processing steps (Garson et al. (2009)), a standardized approach to each step of the RT-qPCR workflow is critical for reliable and reproducible results. The MIQE provides this approach with a checklist that contains 85 parameters to assure quality results that will meet the acceptance criteria of any journal (Bustin et al. (2009)). In this paper we demonstrate how to apply the MIQE guidelines (www.rdml.org/miqe) to establish a solid experimental approach.

Molecular mechanisms of metastasis.

A major topic covered at the First International Symposium on Cancer Metastasis and the Lymphovascular System was the molecular mechanisms of metastasis. This has become of major interest in recent years as we have discovered new metastasis-related genes and gained understanding of the molecular events of lymphatic metastasis. The symposium covered new aspects and important questions related to the events of metastasis in both humans and animals. The basic and clinical related research covered in this topic represented many disciplines. The presentations showed novel findings and at the same time, raised many new unanswered questions, indicating the limited knowledge we still have regarding the molecular events of metastasis. The hope is that further unraveling of the direct and indirect molecular events of lymphatic metastasis will lead to new approaches in developing effective therapeutics.

A GM-CSF/CD40L producing cell augments anti-tumor T cell responses.

BACKGROUND: Tumors evade T cell-mediated rejection despite the presence of tumor associated antigens (TAAs) and T cells specific for these TAAs in cancer patients. Therapeutic tumor vaccines are being developed to prevent this evasion. Previous reports revealed that anti-tumor T cell responses could be activated in mice when granulocyte macrophage-colony stimulating factor (GM-CSF) or CD40L are produced at tumor vaccine sites. We sought to test the hypothesis that production of GM-CSF and CD40L by a bystander cell line could induce an anti-tumor T cell response in an in vitro human model. MATERIALS AND METHODS: The K562 cell line was stably transfected with the human GM-CSF and CD40L genes. The effect of this cell line on T cell responses was tested in a human autologous mixed tumor cell/lymph node cell model using tissue from a series of cancer patients. RESULTS: There was no significant anti-tumor T cell response when human lymphocytes derived from tumor-draining lymph nodes were stimulated with autologous tumor cells in vitro. However, significant anti-tumor T cell responses were observed when bystander cells transfected with CD40L and GM-CSF were added to the cultures. CONCLUSIONS: A fully autologous human model consisting of tumor cells as stimulator cells and tumor-draining lymph nodes as responder cells can be used to test immunotherapeutic strategies. T cells in these lymph nodes are unresponsive to autologous tumor cells, but this lack of responsiveness can be reversed in the presence of GM-CSF and CD40L. These data provide a rationale for testing tumor cell vaccines incorporating GM-CSF- and CD40L-expressing bystanders in clinical trials.

Correlation of osteopontin protein expression and pathological stage across a wide variety of tumor histologies.

PURPOSE: Osteopontin (OPN) is an integrin-binding protein overexpressed in various experimental models of malignancy and appears to be involved in tumorigenesis and metastasis. Although various studies have assessed OPN protein levels in several tumor types, a broad survey of OPN expression in human neoplasia under the same experimental conditions has not been carried out. EXPERIMENTAL DESIGN: We used immunohistochemistry to detect OPN in a selection of 350 human tumors and 113 normal tissues, from a variety of body sites, using stage-oriented human cancer tissue arrays. Tumors included malignancies from breast (26), ovary (22), endometrium (14), esophagus (10), stomach (11), pancreas (16), bile duct (1), liver (9), colon (20), kidney (53), bladder (33), prostate (28), head and neck (60), salivary glands (14), lung (17), skin (6), and brain (10). RESULTS: High cytoplasmic OPN staining was observed in 100% of gastric carcinomas, 85% of colorectal carcinomas, 82% of transitional cell carcinomas of the renal pelvis, 81% of pancreatic carcinomas, 72% of renal cell carcinomas, 71% of lung and endometrial carcinomas, 70% of esophageal carcinomas, 58% of squamous cell carcinomas of the head and neck, and 59% of ovarian carcinomas. Although OPN expression was identified in a good number of bladder, prostate, and brain tumors, the majority of 6 skin cancers, 11 of 14 salivary gland cancers, 2 thyroid carcinomas, and 23 of 26 breast cancers revealed low OPN positivity or were negative. When considering all sites, OPN expression significantly correlated with tumor stage (Spearman's correlation coefficient, P = 0.0002). OPN score and stage were also significantly correlated for specific cancer sites including bladder (P = 0.01), colon (P = 0.004), kidney (P = 0.0001), larynx (P = 0.035), mouth (P = 0.046), and salivary gland (P = 0.011). CONCLUSIONS: This study reports the broad distribution of OPN in human tumors from different body sites, suggesting involvement of this protein in tumor formation. The strong correlation between pathological stage and OPN across multiple tumor types suggests a role for OPN in tumor progression.

Indoleamine 2,3-dioxygenase contributes to tumor cell evasion of T cell-mediated rejection.

The priming of an appropriate anti-tumor T cell response rarely results in the rejection of established tumors. The characteristics of tumors that allow them to evade a T cell-mediated rejection are unknown for many tumors. We report on evidence that the expression of the immunosuppressive enzyme, indoleamine 2,3-dioxygenase (IDO) by mononuclear cells that invade tumors and tumor-draining lymph nodes, is 1 mechanism that may account for this observation. Lewis lung carcinoma (LLC) cells stimulated a more robust allogeneic T cell response in vitro in the presence of a competitive inhibitor of IDO, 1-methyl tryptophan. When administered in vivo this inhibitor also resulted in delayed LLC tumor growth in syngeneic mice. Our study provides evidence for a novel mechanism whereby tumors evade rejection by the immune system, and suggests the possibility that inhibiting IDO may be developed as an anti-cancer immunotherapeutic strategy.

Phase I trial of a B7-1 (CD80) gene modified autologous tumor cell vaccine in combination with systemic interleukin-2 in patients with metastatic renal cell carcinoma.

PURPOSE: A reason that the immune system may fail to reject tumors is that T cells encounter tumor antigen derived peptides on the surface of tumor cells in a tolerizing rather than activating context since tumor cells do not express T cell co-stimulatory molecules such as B7-1 (CD80). In preclinical models over expression of B7-1 on the surface of tumor cells has been shown to activate T cells which kill tumor cells. We conducted a phase I clinical trial testing this approach in patients with metastatic renal cell carcinoma. MATERIALS AND METHODS: Resected tumors from 15 patients were disaggregated and adapted to tissue culture, transduced with the B7-1 gene and injected subcutaneously as a vaccine. The dose of the vaccine was escalated in 3 separate cohorts of patients, and systemic interleukin-2 (IL-2) was administered as an adjuvant designed to enhance the proliferation of the vaccine activated T cells. RESULTS: Of the 15 patients 9 had measurable disease, 2 had a partial response and 2 had stable disease. Perivascular T cell infiltrates at autologous tumor delayed type hypersensitivity skin test sites developed in 3 of the 4 patients with stable disease or partial response. Although the patients experienced the usual and expected toxicity from the IL-2, there was no significant toxicity observed with the vaccine. CONCLUSIONS: The B7-1 gene modified autologous tumor cell vaccine is safe and can be combined with systemic IL-2 with acceptable toxicity. Immunological and clinical responses were observed in some of the patients. A phase II trial is reasonable to determine the efficacy of this approach.