Noriko Sakemura

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 GPC3144–152 (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 GPC3144–152 peptide vaccine in 14 patients with advanced HCC. Immunological responses were analyzed by ex vivoγ‐interferon enzyme‐linked immunospot assay. The frequency of GPC3144–152 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 GPC3144–152 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 GPC3144–152 peptide‐specific CTLs after vaccination and the dose of the peptide injected (P = 0.0166, r = 0.665). Moreover, we established several GPC3144–152 peptide‐specific CTL clones from PBMCs of patients vaccinated with GPC3144–152 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 GPC3144–152 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. (Cancer Sci 2011; 102: 918–925)

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

Abstract:  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.

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.

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

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #4150 Backgrounds: Breast-conserving surgery (BCS) is a standard care for early breast cancer patients. However, local control is not sufficient for surgery alone and breast irradiation and hormonal treatment in hormone-sensitive tumor are recommended to reduce 10% or less of local failure rate. To evaluate the safety and reliability of thermal ablation therapy instead of BCS, we performed radiofrequency ablation (RFA) for clinical stage I breast cancer patients. Patients and methods: The subject was T1N0 breast cancer patient with no extensive intraductal components. Under general anesthesia, sentinel node biopsy, and then RFA immediately followed by BCS was performed. Resected specimens were examined at 5 mm intervals by hematoxylin-eosin (HE) staining and several slices of tumor and non tumor lesions in each case were also evaluated by nicotinamide adenine dinucleotide (NADH) diaphorase staining. Results: Thirty of the 34 eligible patients were enrolled (88%) about 2 years. Tumor diameters by ultrasound ranged from 9 mm to 24 mm. RFA-relating adverse events were observed in 2 cases of skin burn and 7 of muscle burn in major pectral muscle. There was one case of technical failure due to insufficient electrode replacement into the tumor. Twenty-eight cases (93%) revealed pathological degeneration changes of tumor specimens with HE staining. However, 2 cases had viable tumor cells of intraductal components beyond ablated breast tissues. In 24 of the 26 cases (92%) examined by NADH diphorase staining, tumor viability was diagnosed as negative. Finally, the complete ablation rate was 87% (26/30). Conclusions: RFA proved to be reliable and feasible in clinical stage I breast cancer without extensive intraductal components. RFA will be an alternative to BCS as surgical treatment in such a case. Randomized clinical trials to compare RFA with BCS in breast cancer should be required. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4150.

Abstract 3508: Zoledronate induce expansion of glypican-3 (GPC3) peptide specific cytotoxic T lLymphocytes sufficient for adoptive cancer immunotherapy

BACKGROUND Glypican-3 (GPC3) is an onco-fetal antigen which is overexpressed in human hepatocellular carcinoma (HCC), and is only expressed in the placenta and embryonic liver among normal tissues. Previously, we completed a phase I clinical trial of HLA-A2-restricted GPC3 144-152 (FVGEFFTDV) and A24-restricted GPC3 298-306 (EYILSLEEL) peptide vaccine in 33 patients with advanced HCC. This clinical trial of GPC3-derived peptide vaccine showed the vaccination to be safe, and indicates many immunological responses. To develop more efficient immunotherapy, we investigated large scale expansion of GPC3 peptide specific CTLs for adoptive immunotherapy. PURPOSE In this study, we investigated the efficiency of new method to induce expansion of GPC3 peptide specific CTLs. METHODS Treatment of human peripheral blood mononuclear cells (PBMCs) with zoledronate is a method that enables large-scale γδ T cell expansion. To induce expansion of GPC3 peptide specific CTLs and γδ T cells, the PBMCs of patients vaccinated with GPC3 peptide were cultured with GPC3 peptide and zoledronate for 14 days. We used CD8 + and CD8 − cells that isolated from cultured cells using CD8 microbeads at day 14 as a effector cells. GPC3 peptide specificity and cytotoxic activity of CTLs were analysed by Dextramer assay, cytotoxicity assay and IFN-γ ELISPOT assay. We used human liver cancer cell line SK-Hep-1 (GPC3 − , HLA-A*02:01/24:02) and a human GPC3 gene transfectant, SK-Hep-1 [[Unsupported Character - ⁄]] hGPC3 (GPC3 + , HLA-A*02:01/24:02) as target cells. T2 (HLA-A*02:01, TAP − ) was pulsed with GPC3 peptide or HIV peptide at room temperature for 1h. To evaluate antitumor activity in vivo, we inoculated SK-Hep-1/hGPC3 and SK-Hep-1/vec cells at the right flank of NOD/SCID mice. We injected the CD8 + , CD8 − or all cells as effector cells intravenously. RESULTS The expansion of Lymphocytes from a few PBMCs of vaccinated patients with advanced HCC using zoledronate yields cell numbers sufficient for adoptive transfer. The rate of increase of GPC3 specific CTLs was approximate 490 to 170,000-fold, whereas the rate of increase of total cell number was approximate 13 to 1,000-fold. These CD8 + cells including CTLs showed GPC3 specific cytotoxicity against SK-Hep-1/hGPC3 and T2 pulsed with GPC3 peptide, but not against SK-Hep-1/vec and T2 pulsed with HIV peptide, whereas CD8 − cells including γδ T cells showed cytotoxicity against SK-Hep-1/hGPC3 and SK-Hep-1/vec while did not show GPC3 specificity. Furthermore, adoptive cell transfer of CD8 + cells, CD8 − cells and total cells after expansion significantly inhibited tumor growth in NOD/SCID mouse model. CONCLUSION This study indicates that zoledronate is useful to large-scale expand antigen-specific CTLs and γδ T cells for adoptive immunotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3508. doi:1538-7445.AM2012-3508

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

BackgroundSentinel 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.MethodsRecent 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.ResultsITC 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.ConclusionsSNB 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.

Abstract 5439: A novel drug sensitivity assay method for 3D cultured cell using an imaging device

Three-dimensional (3D) cultured cells are useful for the physiological study of tumor cells owing to their similar characteristics. Currently, the popular methods for 3D cell culture-for example, collagen gel or the matrix method-involve complicated procedures that often result in experimental errors. We attemted to develop a novel technique for the use of 3D cell culture in a novel drug sensitivity assay using an imaging technique. We used a NanoCulture® plate (NCP) for 3D cell culture of the human breast cancer cell lines BT474 and T47D for forming multicellular spheroids and used these in a drug sensitivity assay for trastuzumab and paclitaxel by employing the imaging device BioStation CT. The NCP has a fine pattern carved on the surface of the wells’ bottoms, which provides scaffolding for cells with expanding pseudopodia. Thus, the cells can move and aggregate to form uniform spheroids on the NCP. These spheroids further migrate and fuse each other during culture. The effects of anticancer drugs-i.e., a decrease in the migration velocity of spheroids and suppression of spheroid fusion in a dose-dependent manner-were estimated using NCP-cultures spheroids analyzed using sequential BioStation CT images. These results were compared to the conventional assay method of ATP quantification with a good agreement. We confirmed the antitumor effect of the drugs on cells seeded in a single well of the 96-well plate by this technique. We expect this method to be useful in research on new antitumor agents and in drug sensitivity tests for individually tailored cancer treatments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5439. doi:10.1158/1538-7445.AM2011-5439

Abstract 2688: Establishment of high avidity CTL clones induced by HLA-A2 restricted glypican-3 (GPC3) peptide vaccine

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Glypican-3 (GPC3) is an onco-fetal antigen which is overexpressed in human hepatocellular carcinoma (HCC) and that among normal tissues it is slightly expressed in placenta and embryonic liver. Previously, we have identified a HLA-A2-restricted GPC3144-152 (FVGEFFTDV) peptide which can induce GPC3-reactive cytotoxic T lymphocytes (CTLs) without inducing autoimmunity. In the present study, we performed phase I clinical trial of HLA-A2 restricted GPC3-derived peptide vaccine to the 14 patients with advanced HCC. Immunological responses were analyzed by ex vivo IFN-γ Enzyme-linked immunospot (ELISPOT) assay. The frequency of GPC3 peptide specific CTLs after vaccinations (mean, 96; range, 5-441) was statistically significantly larger than that before vaccination (mean, 6.5; range, 0-43) (P < 0.01). An increase of the GPC3 peptide specific CTL frequency was detected in 12 (86%) of 14 patients after vaccination. Additionally, there was a significant correlation between the maximum value of GPC3 peptide specific CTLs after vaccination and dose of peptide injected (P = 0.0166, r = 0.665). Moreover, we established some GPC3 peptide specific CTL clones from PBMCs of patients vaccinated with GPC3 peptide by single cell sort using Dextramer and CD107a antibody. These CTL clones were high avidity CTLs (10−10M and 10−11M) and could recognize HCC cell lines expressing GPC3 in a HLA-class I-restricted manner. These results suggest that tumor-cytolytic T cells are indeed elicited in patients after vaccination. Now we investigate whether the GPC3 based immunotherapy have possibility of the application for other GPC3 expressing cancers including melanoma, clear cell carcinoma of the ovary, pediatric cancer or not, using established GPC3 peptide specific CTL clone. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2688. doi:10.1158/1538-7445.AM2011-2688

Abstract 1555: Immune suppression of regulatory T cells and M2 macrophage in breast cancer patients

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: Recent multi-disciplinary treatments of breast cancer (BC) could decrease the mortality rate, but successful immune therapy remains uncertain. To explore new strategy of immune therapy of BC, we are investigating about host-tumor immune response in BC patients. Materials and methods: Tumor tissue specimens and peripheral blood mononuclear cells (PBMC) were obtained from early and advanced BC patients. PBMC were also collected from healthy volunteers. Regulatory T (Treg) cells were examined by counting CD4+CD25highCD127low/-cells in PBMC with flow cytometry analysis. Immunohistochemical evaluation of tumor specimens was performed with monoclonal antibodies of HLA-ABC and DR, CD56, CD68, CD83, and CD163. The number of stained cells was analyzed using a semiquantitative ordinal scale ranging from 0 to 3 (0, +/-, ++, +++). Results: HLA type I and type II were stained negative in 44% and 82% of 50 BC cases. When host-tumor immune response were compared between 38 early BC cases and 12 advanced BC cases, numbers of CD68-positive cells significantly increased in peripheral tumor tissues of advanced BC cases than in those of early BC cases (92% versus 53%). CD163-positive tumor cells were also detected more frequently in advanced BC cases than in early BC cases (75% versus 16%). There were no significant differences of distribution of CD4, CD8, CD56, and CD83-positive cells in early and advanced BC cases. Treg cells in PBMC significantly increased in percentage of the population in 46 BC patients than in 16 healthy volunteers (3.8% versus 2.1% of CD4-positive cells at mean value). Interestingly, Treg cells decreased in percentage of the population in postoperative BC patients. Conclusions: Our results suggest that Treg cells render BC patients under immune suppression and M2 macrophage plays an important role of tumor progression. Targeted therapy against M2 macrophage traits may be a promising strategy of breast cancer. Host-tumor immune response will be reported for BC patients treated with primary chemotherapy or radiofrequency ablation therapy in comparison with those who underwent breast surgery first. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1555. doi:10.1158/1538-7445.AM2011-1555