Intratumoral peptide injection enhances tumor cell antigenicity recognized by cytotoxic T lymphocytes: a potential option for improvement in antigen-specific cancer immunotherapy.

Antigen-specific cancer immunotherapy is a promising strategy for improving cancer treatment. Recently, many tumor-associated antigens and their epitopes recognized by cytotoxic T lymphocytes (CTLs) have been identified. However, the density of endogenously presented antigen-derived peptides on tumor cells is generally sparse, resulting in the inability of antigen-specific CTLs to work effectively. We hypothesize that increasing the density of an antigen-derived peptide would enhance antigen-specific cancer immunotherapy. Here, we demonstrated that intratumoral peptide injection leads to additional peptide loading onto major histocompatibility complex class I molecules of tumor cells, enhancing tumor cell recognition by antigen-specific CTLs. In in vitro studies, human leukocyte antigen (HLA)-A*02:01-restricted glypican-3144-152 (FVGEFFTDV) and cytomegalovirus495-503 (NLVPMVATV) peptide-specific CTLs showed strong activity against all peptide-pulsed cell lines, regardless of whether the tumor cells expressed the antigen. In in vivo studies using immunodeficient mice, glypican-3144-152 and cytomegalovirus495-503 peptides injected into a solid mass were loaded onto HLA class I molecules of tumor cells. In a peptide vaccine model and an adoptive cell transfer model using C57BL/6 mice, intratumoral injection of ovalbumin257-264 peptide (SIINFEKL) was effective for tumor growth inhibition and survival against ovalbumin-negative tumors without adverse reactions. Moreover, we demonstrated an antigen-spreading effect that occurred after intratumoral peptide injection. Intratumoral peptide injection enhances tumor cell antigenicity and may be a useful option for improvement in antigen-specific cancer immunotherapy against solid tumors.

Paradigm shift in axilla surgery for breast cancer patients treated with sentinel node biopsy.

BACKGROUND: Sentinel node biopsy (SNB) is a standard technique for the diagnosis of regional lymph node metastases in clinically node-negative breast cancer patients. In the case of pathologically negative sentinel lymph nodes (SLN), axillary lymph node dissection (ALND) can be avoided. METHODS: Recent clinical studies on SNB in breast cancer were reviewed regarding the pathological and molecular diagnosis of SLN, the tools used to predict non-SLN metastases, the prognostic significance of isolated tumor cells (ITC) and micrometastases (MIC), and axilla surgery. RESULTS: ITC or MIC in SLN was associated with worse survival in patients treated with SNB alone or SNB followed by ALND. However, this effect was limited and adjuvant therapy improved survival. If T1 and one SLN-positive breast cancer patients are treated with whole-breast irradiation and adjuvant therapy, additional ALND may not be necessary. CONCLUSIONS: SNB without ALND can be adopted for patients with a small number of SLN metastases. Although the lack of apparent regional lymph node recurrence, similar to tumor dormancy, cannot be fully explained, ALND should be performed in cases that are highly suspected to be non-SLN metastases.

Identification of an HLA-A*0201-restricted cytotoxic T lymphocyte epitope from the lung carcinoma antigen, Lengsin.

Lengsin is an eye lens protein with a glutamine synthetase domain. We previously identified this protein as a lung carcinoma antigen through cDNA microarray analysis. Lengsin protein is overexpressed irrespective of the histological type of lung carcinoma, but not in normal tissues other than the lens. Therefore, to significantly extend the use of Lengsin-based T-cell immunotherapies for the treatment of patients with lung carcinoma, we searched for HLA-A*0201-restricted epitopes from this protein by screening predicted Lengsin-derived candidate peptides for the induction of tumor-reactive CTLs. Four Lengsin-derived peptides were selected by computerized algorithm based on a permissive HLA-A*0201 binding motif, and were used to immunize HLA-A*0201 transgenic (HHD) mice. Two of the immunizing peptides, Lengsin(206-215)(FIYDFCIFGV) and Lengsin(270-279)(FLPEFGISSA), induced peptide-specific cytotoxic T lymphocytes (CTLs) in HHD mice, and thus were used to stimulate human peripheral blood lymphocytes in vitro. Lengsin(206-215) and Lengsin (270-279) also induced human peptide-specific CTLs, and we were able to generate Lengsin(206-215)- and Lengsin(270-279)-specific CTL clones. The Lengsin(270-279)-specific CTL clone specifically recognized peptide-pulsed T2 cells, COS-7 cells expressing HLA-A*0201 and Lengsin, and HLA-A*0201+/Lengsin+ lung carcinoma cells in an HLA-A*0201-restricted manner. On the other hand, the Lengsin(206-215)-specific CTL clone failed to recognize HLA-A*0201+/Lengsin+ target cells in the absence of cognate peptide. These results suggest that Lengsin(270-279) is naturally processed and presented by HLA-A*0201 molecules on the surface of lung carcinoma cells and may be a new target for antigen-specific T-cell immunotherapy against lung cancer.

HLA-A2-restricted glypican-3 peptide-specific CTL clones induced by peptide vaccine show high avidity and antigen-specific killing activity against tumor cells.

Glypican-3 (GPC3) is an onco-fetal antigen that is overexpressed in human hepatocellular carcinoma (HCC), and is only expressed in the placenta and embryonic liver among normal tissues. Previously, we identified an HLA-A2-restricted GPC3(144-152) (FVGEFFTDV) peptide that can induce GPC3-reactive CTLs without inducing autoimmunity in HLA-A2 transgenic mice. In this study, we carried out a phase I clinical trial of HLA-A2-restricted GPC3(144-152) peptide vaccine in 14 patients with advanced HCC. Immunological responses were analyzed by ex vivo γ-interferon enzyme-linked immunospot assay. The frequency of GPC3(144-152) peptide-specific CTLs after vaccination (mean, 96; range, 5-441) was significantly larger than that before vaccination (mean, 6.5; range, 0-43) (P < 0.01). An increase in the GPC3(144-152) peptide-specific CTL frequency was observed in 12 (86%) of 14 patients after vaccination. Additionally, there was a significant correlation between the maximum value of GPC3(144-152) peptide-specific CTLs after vaccination and the dose of the peptide injected (P = 0.0166, r = 0.665). Moreover, we established several GPC3(144-152) peptide-specific CTL clones from PBMCs of patients vaccinated with GPC3(144-152) peptide by single cell sorting using Dextramer and CD107a antibody. These CTL clones had high avidity (the recognition efficiency showing 50% cytotoxicity was 10(-10) or 10(-11) M) and could recognize HCC cell lines expressing GPC3 in an HLA-class I-restricted manner. These results suggest that GPC3(144-152) peptide vaccine can induce high avidity CTLs capable of killing HCC cells expressing GPC3. This trial was registered with University Hospital Medical Information Network number 000001395.

Feasibility study on radiofrequency ablation followed by partial mastectomy for stage I breast cancer patients.

To evaluate the safety and reliability of thermal ablation therapy instead of breast-conserving surgery (BCS), we performed radiofrequency ablation (RFA) for clinical stage I breast cancer patients. Subjects were T1N0 breast cancer patients with no extensive intraductal components. Under general anesthesia, sentinel node biopsy was performed, followed by RFA and BCS. Resected specimens were examined at 5-mm intervals by hematoxylin-eosin (H&E) staining and nicotinamide adenine dinucleotide (NADH) diaphorase staining. Thirty of the 34 eligible patients were enrolled. RFA-related adverse events were observed in nine patients: two with skin burn and seven with muscle burn. Twenty-six patients (87%) showed pathological degenerative changes in tumor specimens with H&E staining. In 24 of the 26 cases (92%) examined by NADH diaphorase staining, tumor cell viability was diagnosed as negative. RFA proved to be reliable and feasible in clinical stage I breast cancer, with no extensive intraductal components. Randomized clinical trials are needed to compare RFA with BCS.

Invasive apocrine carcinoma of the breast: clinicopathologic features of 57 patients.

Apocrine carcinoma is a rare, unique, and morphologically distinctive type of invasive ductal carcinoma (IDC). The features of invasive apocrine carcinoma (IAC) and their possible prognostic implications have not been fully investigated. To this end, we examined the clinicopathologic characteristics and outcome of patients with IAC and compared these factors with those of patients with IDC. Out of 2,055 breast cancer patients who had undergone breast surgery between 1995 and 2005, 57 patients of IAC and 1,583 patients of IDC were analyzed. The mean ages of the patients with IAC and of those with IDC were 58.5 +/- 10.9 years and 54.4 +/- 11 years, respectively (p = 0.006). The percentages of patients with axillary nodal metastasis and lymphatic invasion were significantly lower in the IAC group than in the IDC group (p = 0.03 and 0.02, respectively). The percentage of estrogen and progesterone receptor negativity was higher in the IAC group than in the IDC group (p < 0.001). After a median follow-up period of 49 months (range, 1-133 months), seven (12%) patients with IAC and 244 (15%) patients with IDC had experienced recurrences. Three (5%) patients with IAC and 125 (8%) patients with IDC died of recurrent breast cancer. No significant differences in the relapse-free survival (p = 0.83) and overall survival (p = 0.75) rates were observed between the two groups. Although IAC and IDC have different clinicopathologic characteristics, the prognoses of patients with these diseases are similar.